Novel anti-cd38 antibodies for the treatment of cancer

ABSTRACT

Antibodies, humanized antibodies, resurfaced antibodies, antibody fragments, derivatized antibodies, and conjugates of same with cytotoxic agents, which specifically bind to CD38, are capable of killing CD38 +  cells by apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and/or complement-dependent cytotoxicity (CDC). Said antibodies and fragments thereof may be used in the treatment of tumors that express CD38 protein, such as multiple myeloma, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, or acute lymphocytic leukemia, or the treatment of autoimmune and inflammatory diseases such as systemic lupus, rheumatoid arthritis, multiple sclerosis, erythematosus, and asthma. Said derivatized antibodies may be used in the diagnosis and imaging of tumors that express elevated levels of CD38. Also provided are cytotoxic conjugates comprising a cell binding agent and a cytotoxic agent, therapeutic compositions comprising the conjugate, methods for using the conjugates in the inhibition of cell growth and the treatment of disease, and a kit comprising the cytotoxic conjugate. In particular, the cell binding agent is a monoclonal antibody, and epitope-binding fragments thereof, that recognizes and binds the CD38 protein.

BACKGROUND OF THE INVENTION

CD38 is a 45 kD type II transmembrane glycoprotein with a longC-terminal extracellular domain and a short N-terminal cytoplasmicdomain. The CD38 protein is a bifunctional ectoenzyme that can catalyzethe conversion of NAD⁺ into cyclic ADP-ribose (cADPR) and also hydrolyzecADPR into ADP-ribose. During ontogeny, CD38 appears on CD34⁺ committedstem cells and lineage-committed progenitors of lymphoid, erythroid andmyeloid cells. CD38 expression persists mostly in the lymphoid lineagewith varying expression levels at different stages of T and B celldevelopment.

CD38 is upregulated in many hematopoeitic malignancies and in cell linesderived from various hematopoietic malignancies, including non-Hodgkin'slymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), Bchronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia(ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cellleukemia (HCL), Hodgkin's Lymphoma (HL), and chronic myeloid leukemia(CML). On the other hand, most primitive pluripotent stem cells of thehematopoietic system are CD38⁻. CD38 expression in hematopoieticmalignancies and its correlation with disease progression makes CD38 anattractive target for antibody therapy.

CD38 has been reported to be involved in Ca²⁺ mobilization (M. Morra etal., 1998, FASEB J., 12: 581-592; M. T. Zilber et al., 2000, Proc NatlAcad Sci USA, 97: 2840-2845) and in the signal transduction throughtyrosine phosphorylation of numerous signaling molecules, includingphospholipase C-γ, ZAP-70, syk, and c-cbl, in lymphoid and myeloid cellsor cell lines (A. Funaro et al., 1993, Eur J Immunol, 23: 2407-2411; M.Morra et al., 1998, FASEB J., 12: 581-592; A. Funaro et al., 1990, JImmunol, 145: 2390-2396; M. Zubiaur et al., 1997, J Immunol, 159:193-205; S. Deaglio et al., 2003, Blood 102: 2146-2155; E. Todisco etal., 2000, Blood, 95: 535-542; M. Konopleva et al., 1998, J Immunol,161: 4702-4708; M. T. Zilber et al., 2000, Proc Natl Acad Sci USA, 97:2840-2845; A. Kitanaka et al., 1997, J Immunol, 159: 184-192; A.Kitanaka et al., 1999, J Immunol, 162: 1952-1958; R. Mallone et al.,2001, Int Immunol, 13: 397-409). On the basis of these observations,CD38 was proposed to be an important signaling molecule in thematuration and activation of lymphoid and myeloid cells during theirnormal development.

The exact role of CD38 in signal transduction and hematopoiesis is stillnot clear, especially since most of these signal transduction studieshave used cell lines ectopically overexpressing CD38 and anti-CD38monoclonal antibodies, which are non-physiological ligands. Because theCD38 protein has an enzymatic activity that produces cADPR, a moleculethat can induce Ca²⁺ mobilization (H. C. Lee et al., 1989, J Biol Chem,264: 1608-1615; H. C. Lee and R. Aarhus, 1991, Cell Regul, 2: 203-209),it has been proposed that CD38 ligation by monoclonal antibodiestriggers Ca²⁺ mobilization and signal transduction in lymphocytes byincreasing production of cADPR (H. C. Lee et al., 1997, Adv Exp MedBiol, 419: 411-419). Contrary to this hypothesis, the truncation andpoint-mutation analysis of CD38 protein showed that neither itscytoplasmic tail nor its enzymatic activity is necessary for thesignaling mediated by anti-CD38 antibodies (A. Kitanaka et al., 1999, JImmunol, 162: 1952-1958; F. E. Lund et al., 1999, J Immunol, 162:2693-2702; S. Hoshino et al., 1997, J Immunol, 158, 741-747).

The best evidence for the function of CD38 comes from CD38⁻/⁻ knockoutmice, which have a defect in their innate immunity and a reduced T-celldependent humoral response due to a defect in dendritic cell migration(S. Partida-Sanchez et al., 2004, Immunity, 20: 279-291; S.Partida-Sanchez et al., 2001, Nat Med, 7: 1209-1216). Nevertheless, itis not clear if the CD38 function in mice is identical to that in humanssince the CD38 expression pattern during hematopoiesis differs greatlybetween human and mouse: a) unlike immature progenitor stem cells inhumans, similar progenitor stem cells in mice express a high level ofCD38 (T. D. Randall et al., 1996, Blood, 87: 4057-4067; R. N. Dagher etal., 1998, Biol Blood Marrow Transplant, 4: 69-74), b) while during thehuman B cell development, high levels of CD38 expression are found ingerminal center B cells and plasma cells (F. M. Uckun, 1990, Blood, 76:1908-1923; M. Kumagai et al., 1995, J Exp Med, 181: 1101-1110), in themouse, the CD38 expression levels in the corresponding cells are low (A.M. Oliver et al., 1997, J Immunol, 158: 1108-1115; A. Ridderstad and D.M. Tarlinton 1998, J Immunol, 160: 4688-4695).

Several anti-human CD38 antibodies with different proliferativeproperties on various tumor cells and cell lines have been described inthe literature. For example, a chimeric OKT10 antibody with mouse Faband human IgG1 Fc mediates antibody-dependent cell-mediated cytotoxicity(ADCC) very efficiently against lymphoma cells in the presence ofperipheral blood mononuclear effector cells from either MM patients ornormal individuals (F. K. Stevenson et al. 1991, Blood, 77: 1071-1079).A CDR-grafted humanized version of the anti-CD38 antibody AT13/5 hasbeen shown to have potent ADCC activity against CD38-positive cell lines(U.S. Ser. No. 09/797,941 A1). Human monoclonal anti-CD38 antibodieshave been shown to mediate the in vitro killing of CD38-positive celllines by ADCC and/or complement-dependent cytotoxicity (CDC), and todelay the tumor growth in SCID mice bearing MM cell line RPMI-8226(WO2005/103083 A2). On the other hand, several anti-CD38 antibodies,IB4, SUN-4B7, and OKT10, but not IB6, AT1, or AT2, induced theproliferation of peripheral blood mononuclear cells (PBMC) from normalindividuals (C. M. Ausiello et al. 2000, Tissue Antigens, 56: 539-547).

Some of the antibodies of the prior art have been shown to be able totrigger apoptosis in CD38⁺ B cells. However, they can only do so in thepresence of stroma cells or stroma-derived cytokines. An agonisticanti-CD38 antibody (IB4) has been reported to prevent apoptosis of humangerminal center (GC) B cells (S. Zupo et al. 1994, Eur J Immunol, 24:1218-1222), and to induce proliferation of KG-1 and HL-60 AML cells (M.Konopleva et al. 1998, J Immunol, 161: 4702-4708), but induces apoptosisin Jurkat T lymphoblastic cells (M. Morra et al. 1998, FASEB J, 12:581-592). Another anti-CD38 antibody T16 induced apoptosis of immaturelymphoid cells and leukemic lymphoblast cells from an ALL patient (M.Kumagai et al. 1995, J Exp Med, 181: 1101-1110), and of leukemicmyeloblast cells from AML patients (E. Todisco et al. 2000, Blood, 95:535-542), but T16 induced apoptosis only in the presence of stroma cellsor stroma-derived cytokines (IL-7, IL-3, stem cell factor).

On the other hand, some prior art antibodies induce apoptosis aftercross-linking, but are totally devoid of any apoptotic activity whenincubated alone (WO 2006/099875).

Because CD38 is an attractive target for antibody therapy for varioushematopoietic malignancies, we generated and screened a large number ofanti-human CD38 antibodies for high potency in the following threecytotoxic activities against CD38-positive malignant hematopoieticcells: induction of apoptosis, ADCC, and CDC. The present inventiondescribes novel anti-CD38 antibodies capable of killing CD38⁺ cells bythree different cytotoxic mechanisms: induction of apoptosis, ADCC, andCDC. Remarkably, the present invention discloses the first anti-CD38antibodies that are able to directly induce apoptosis of CD38⁺ cells,even without the presence of stroma cells or stroma-derived cytokines.

SUMMARY OF THE INVENTION

It is an object of the invention to provide antibodies specificallybinding CD38, and capable of killing CD38⁺ cells by apoptosis. Whereassome prior art antibodies are able to trigger apoptosis only whencrosslinked, but are otherwise devoid of any apoptotic activity, theantibodies of the invention are capable of inducing apoptotic cell deathof CD38+ cells even when incubated alone. In one aspect of theinvention, the antibodies of the invention are capable of killing CD38⁺B cells by ADCC or CDC. In yet another aspect, the antibodies of theinvention are capable of killing CD38⁺ cell by at least two of theaforementioned mechanims, i.e. apoptosis, ADCC, and CDC. Remarkably, theantibodies of the invention are the first anti-CD38 antibodies that havebeen demonstrated to kill CD38⁺ B cells by all three differentmechanisms: apoptosis, ADCC, and CDC. In a further embodiment of theinvention, said antibodies are capable of killing CD38⁺ B cells byapoptosis even in the absence of stroma cells or stroma-derivedcytokines.

The antibodies of the invention are capable in particular of killingmalignant CD38⁺ B cells, including lymphoma cells, leukemia cells, andmultiple myeloma cells. In some embodiments, the CD38⁺ B cell is a NHL,BL, MM, B-CLL, ALL, TCL, AML, HCL, HL, or CML cell.

In one aspect of the invention, the antibodies of the invention arecapable of killing at least 24% of Daudi lymphoma cells and/or at least7% of Ramos lymphoma cells and/or 11% of MOLP-8 multiple myeloma cellsand/or 36% of SU-DHL-8 lymphoma cells and/or 62% of DND-41 leukemiacells and/or 27% of NU-DUL-1 lymphoma cells and/or 9% of JVM-13 leukemiacells and/or 4% of HC-1 leukemia cells by apoptosis in the absence ofstroma cells or stroma-derived cytokines.

Antibodies of the invention can be polyclonal or monoclonal. Preferredare monoclonal anti-CD38 antibodies. In a more preferred embodiment,there are provided murine antibodies selected from 38SB13, 38SB18,38SB19, 38SB30, 38SB31, and 38SB39, which are fully characterized hereinwith respect to the amino acid sequences of both their light and heavychain variable regions, the cDNA sequences of the genes for the lightand heavy chain variable regions, the identification of their CDRs(complementarity-determining regions), the identification of theirsurface amino acids, and means for their expression in recombinant form.

The present invention includes chimeric versions of the murine anti-CD38monoclonal antibody selected from 38SB13, 38SB18, 38SB19, 38SB30,38SB31, and 38SB39. Also included are resurfaced or humanized versionsof the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodieswherein surface-exposed residues of the variable region frameworks ofthe antibodies, or their epitope-binding fragments, are replaced in bothlight and heavy chains to more closely resemble known human antibodysurfaces. The humanized antibodies and epitope-binding fragments thereofof the present invention have improved properties in that they are lessimmunogenic (or completely non-immunogenic) than murine versions inhuman subjects to which they are administered. Thus, the differentversions of humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39antibodies and epitope-binding fragments thereof of the presentinvention specifically recognize CD38 while not being immunogenic to ahuman.

The humanized versions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31,and 38SB39 antibodies of the present invention are fully characterizedherein with respect to their respective amino acid sequences of bothlight and heavy chain variable regions, the DNA sequences of the genesfor the light and heavy chain variable regions, the identification ofthe complementarity determining regions (CDRs), the identification oftheir variable region framework surface amino acid residues, anddisclosure of a means for their expression in recombinant form.

This invention also contemplates the use of conjugates between cytotoxicconjugates comprising (1) a cell binding agent that recognizes and bindsCD38, and (2) a cytotoxic agent. In the cytotoxic conjugates, the cellbinding agent has a high affinity for CD38 and the cytotoxic agent has ahigh degree of cytotoxicity for cells expressing CD38, such that thecytotoxic conjugates of the present invention form effective killingagents.

In a preferred embodiment, the cell binding agent is an anti-CD38antibody (e.g., 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39) oran epitope-binding fragment thereof, more preferably a humanizedanti-CD38 antibody (e.g., 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and38SB39) or an epitope-binding fragment thereof, wherein a cytotoxicagent is covalently attached, directly or via a cleavable ornon-cleavable linker, to the antibody or epitope-binding fragmentthereof. In more preferred embodiments, the cell binding agent is thehumanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodiesor an epitope-binding fragment thereof, and the cytotoxic agent is ataxol, a maytansinoid, a tomaymycin derivative, a leptomycin derivative,CC-1065 or a CC-1065 analog.

More preferably, the cell binding agent is the humanized anti-CD38antibody 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39, and thecytotoxic agent is a maytansine compound, such as DM1 or DM4.

The present invention also encompasses the use of fragments of anti-CD38antibodies which retain the ability to bind CD38. In another aspect ofthe invention, the use of functional equivalents of anti-CD38 antibodiesis contemplated.

The present invention also includes a method for inhibiting the growthof a cell expressing CD38. In preferred embodiments, the method forinhibiting the growth of the cell expressing CD38 takes place in vivoand results in the death of the cell, although in vitro and ex vivoapplications are also included.

The present invention also provides a therapeutic composition comprisingan anti-CD38 antibody or an anti-CD38 antibody-cytotoxic agentconjugate, and a pharmaceutically acceptable carrier or excipients. Insome embodiments, the therapeutic composition comprises a secondtherapeutic agent. This second therapeutic agent can be chosen from thegroup comprising the antagonists of epithermal-growth factor (EGF),fibroblast-growth factor (FGF), hepatocyte growth factor (HGF), tissuefactor (TF), protein C, protein S, platelet-derived growth factor(PDGF), heregulin, macrophage-stimulating protein (MSP) or vascularendothelial growth factor (VEGF), or an antagonist of a receptor forepidermal-growth factor (EGF), fibroblast-growth factor (FGF),hepatocyte growth factor (HGF), tissue factor (TF), protein C, proteinS, platelet-derived growth factor (PDGF), heregulin,macrophage-stimulating protein (MSP), or vascular endothelial growthfactor (VEGF), including HER2 receptor, HER3 receptor, c-MET, and otherreceptor tyrosine kinases. This second therapeutic agent can be alsochosen from the group comprising of antibodies targeting clusters ofdifferentiation (CD) antigens, including CD3, CD14, CD19, CD20, CD22,CD25, CD28, CD30, CD33, CD36, CD40, CD44, CD52, CD55, CD59, CD56, CD70,CD79, CD80, CD103, CD134, CD137, CD138, and CD152. This secondtherapeutic agent can be also chosen from the group of chemotherapeuticor immunomodulatory agents.

The present invention further includes a method of treating a subjecthaving a cancer or an inflammatory disease, including autoimmune diseaseusing the therapeutic composition. In some embodiments, the cancer isselected from a group consisting of NHL, BL, MM, B-CLL, ALL, TCL, AML,HCL, HL, and CML. In another embodiment, the autoimmune disease isselected from a group consisting of systemic lupus erythematosus,multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerativecolitis, gastritis, Hashimoto's thyroiditis, ankylosing spondylitis,hepatitis C-associated cryoglobulinemic vasculitis, chronic focalencephalitis, bullous pemphigoid, hemophilia A, membranoproliferativeglomerulnephritis, Sjogren's syndrome, adult and juveniledermatomyositis, adult polymyositis, chronic urticaria, primary biliarycirrhosis, idiopathic thrombocytopenic purpura, neuromyelitis optica,Graves' dysthyroid disease, bullous pemphigoid, membranoproliferativeglonerulonephritis, Churg-Strauss syndrome, and asthma. In preferredembodiments, the cytotoxic conjugate comprises an anti-CD38 antibody anda cytotoxic agent. In more preferred embodiments, the cytotoxicconjugate comprises a humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31,and 38SB39 antibody-DM1 conjugate, humanized 38SB13, 38SB18, 38SB19,38SB30, 38SB31, and 38SB39 antibody-DM4 or a humanized 38SB13, 38SB18,38SB19, 38SB30, 38SB31, and 38SB39 antibody-taxane conjugate, and theconjugate is administered along with a pharmaceutically acceptablecarrier or excipients.

In another aspect of the invention, anti-CD38 antibodies are used todetect the CD38 protein in a biological sample. In a preferredembodiment, said antibodies are used to determine CD38 levels in tumortissue.

The present invention also includes a kit comprising an anti-CD38antibody or an anti-CD38 antibody-cytotoxic agent conjugate andinstructions for use. In preferred embodiments, the anti-CD38 antibodiesare the humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39antibodies, the cytotoxic agent is a maytansine compound, such as DM1 orDM4, a tomaymycin derivative, a leptomycin derivative, or a taxane, andthe instructions are for using the conjugates in the treatment of asubject having cancer. The kit may also include components necessary forthe preparation of a pharmaceutically acceptable formulation, such as adiluent if the conjugate is in a lyophilized state or concentrated form,and for the administration of the formulation.

Unless otherwise stated, all references and patents cited herein areincorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a FACS analysis of the specific binding of purified murineanti-CD38 antibodies, 38SB13, 38SB18, 38SB19, 38 to the 300-19 cellsexpressing human CD38 and CD38-positive Ramos lymphoma cells.

FIG. 1B shows a FACS analysis of the specific binding of purified murineanti-CD38 antibodies, 38SB30, 38SB31, 38SB39 and the control anti-CD38antibody AT13/5 to the 300-19 cells expressing human CD38 andCD38-positive Ramos lymphoma cells.

FIG. 2 shows the binding titration curves of 38SB13, 38SB18, 38SB19,38SB30, 38SB31, and 38SB39 established with Ramos cells.

FIG. 3 shows FACS dot plots (FL4-H; TO-PRO-3 staining; y-axis and FL1-H;Annexin V-FITC staining; x-axis) of Ramos cells undergoing apoptosisafter incubation with 38SB13, 38SB19, or AT13/5 (10 nM) for 24 h.

FIG. 4A shows the average percentages of Ramos cells undergoingapoptosis after a 24-h incubation with 38SB13, 38SB18, 38SB19, 38SB30,38SB31, 38SB39, 38SB7, 38SB23, 1B4, AT13/5, OKT10, or SUN-4B7. Theaverage percentage of Annexin V-positive cells (y-axis; includes bothTO-PRO-3 positive and negative cells) from duplicate samples wereplotted.

FIG. 4B shows the average percentages of Daudi cells undergoingapoptosis after a 24-h incubation with the same set of antibodies as inFIG. 4A.

FIG. 4C shows the average percentages of Molp-8 cells undergoingapoptosis after a 24-h incubation with the same set of antibodies as inFIG. 4A.

FIG. 5A shows a diagram of the human expression vector used to expresshu38SB19-LC.

FIG. 5B shows a diagram of the human expression vector used to expresshu38SB19-HC.

FIG. 5C shows a diagram of the human expression vector used to expressboth hu38SB19LC and hu38SB19HC.

FIG. 6A shows ADCC activities mediated by the antibodies, ch38SB13,ch38SB18, and ch38SB19, towards Ramos cells.

FIG. 6B shows ADCC activities mediated by the antibodies, ch38SB30,ch38SB31, and ch38SB39 towards Ramos cells.

FIG. 7A) shows ADCC activities mediated by the antibodies ch38SB18,ch38SB19, ch38SB31, and non-binding chimeric human IgG1 control antibodytowards LP-1 multiple myeloma cells.

FIG. 7B) compares ADCC activities mediated by the antibodies ch38SB19and murine 38SB19 towards Daudi cells.

FIG. 8A shows ADCC activities mediated by the ch38SB19 antibody and bynon-binding chimeric human IgG1 control antibody towards NALM-6 B-ALLcells.

FIG. 8B shows ADCC activities mediated by the ch38SB19 antibody and bynon-binding chimeric human IgG1 control antibody towards MOLT-4 T-ALLcells.

FIG. 9A shows CDC activities mediated by the antibodies ch38SB13,ch38SB18, ch38SB19, ch38SB30, and ch38SB39 towards Raji-IMG cells.

FIG. 9B shows CDC activities mediated by the antibodies ch38SB19 andch38SB31 towards Raji-IMG cells.

FIG. 10 shows CDC activities mediated by the antibodies ch38SB18,ch38SB19, ch38SB31, and by non-binding chimeric human IgG1 controlantibody towards LP-1 multiple myeloma cells.

FIG. 11A shows CDC activities mediated by the antibodies ch38SB13,cch38SB19, and ch38SB39 towards Daudi cells.

FIG. 11B shows CDC activities mediated by the antibodies ch38SB18 andch38SB30 towards Daudi cells.

FIG. 11C shows CDC activities mediated by the antibodies ch38SB19 andch38SB31 towards Daudi cells

FIG. 12A shows the binding titration curves of ch38SB19, hu38SB19 v1.00,and hu38SB19 v1.20 for binding to Ramos cells.

FIG. 12B shows the binding curves that compare ch38SB19, hu38SB19 v1.00,and hu38SB19 v1.00 for their ability to compete with binding ofbiotinylated murine 38SB19 antibody to Ramos cells.

FIG. 13 shows the average percentages of Daudi cells undergoingapoptosis after 24 h of incubation with ch38SB19, hu38SB19 v1.00, orhu38SB19 v1.20 antibody.

FIG. 14 shows ADCC activities mediated by the antibodies ch38SB19,hu38SB19 v1.00, hu38SB19 v1.20, and by non-binding chimeric human IgG1control antibody towards LP-1 multiple myeloma cells.

FIG. 15A shows CDC activities mediated by antibodies ch38SB19, hu38SB19v1.00, and hu38SB19 v1.20 towards Raji-IMG lymphoma cells.

FIG. 15B shows CDC activities mediated by antibodies ch38SB19, hu38SB19v1.00, and hu38SB19 v1.20 towards LP-1 multiple myeloma cells.

FIG. 15C shows CDC activities mediated by antibodies ch38SB19, hu38SB19v1.00, and hu38SB19 v1.20 towards DND-41 T-cell acute lymphoblasticleukemia cells.

FIG. 16 shows the average percentages of Annexin V positive cells after24 h of incubation with hu38SB19 v1.00 antibody for SU-DHL-8 diffuselarge B cell lymphoma cells, NU-DUL-1 B-cell lymphoma cells, DND-41T-cell acute lymphoblastic leukemia cells, JVM-13 B-cell chroniclymphocytic leukemia cells and HC-1 hairy cell leukemia cells.

FIG. 17 shows the percent survival of SCID mice bearing establisheddisseminated human Ramos tumors. Mice were treated with murine 38SB13,38SB18, 38SB19, 38SB30, 38SB31, or 38SB39 antibody or PBS as indicated.

FIG. 18 shows the percent survival of SCID mice bearing establisheddisseminated human Daudi tumors. Mice were treated with hu38SB19 ormu38SB19 antibody or PBS as indicated.

FIG. 19 shows the mean tumor volume of SCID mice bearing NCI-H929multiple myeloma xenograft tumors. Mice were treated with hu38SB19, anon-binding control IgG1 antibody or PBS as indicated.

FIG. 20 shows the mean tumor volume of SCID mice bearing MOLP-8 multiplemyeloma xenograft tumors. Mice were treated with hu38SB19, mu38SB19, anon-binding control IgG1 antibody or PBS as indicated.

DETAILED DESCRIPTION OF THE INVENTION

New antibodies capable of specifically binding CD38 are herein provided.In particular, the present inventors have discovered novel antibodiesthat specifically bind to CD38 on the cell surface and kill CD38⁺ cellsby apoptosis. In one aspect of the invention, the anti-CD38 antibodiesare also capable of killing a CD38⁺ cell by antibody-dependentcytotoxicity (ADCC). In another aspect, the anti-CD38 antibodies of theinvention are capable of killing a CD38⁺ cell by complement-dependentcytotoxicity (CDC). In yet another aspect, the anti-CD38 antibodies ofthe invention are capable of killing a CD38⁺ cell by at least two of theabove mentioned mechanisms, apoptosis, ADCC, and CDC. In particular, ina preferred embodiment, the anti-CD38 antibodies of the invention arecapable of killing a CD38⁺ cell by apoptosis, ADCC, and CDC. Theinvention thus provides the first anti-CD38 antibodies capable ofkilling a CD38⁺ cell by three different mechanisms.

Antibodies capable of binding CD38 and triggering apoptotic cell deathin CD38⁺ cells have been previously described (M. Kumagai et al., 1995,J Exp Med, 181: 1101-1110; E. Todisco et al. 2000, Blood, 95: 535-542),but the antibodies of the invention are the first for which an apoptoticactivity in the absence of stroma cells or stroma-derived cytokines isdemonstrated. The term “stroma” as used herein refers to thenonmalignant supporting tissue of a tumor which includes connectivetissue, blood vessels, and inflammatory cells. Stromal cells producegrowth factors and other substances, including cytokines, that caninfluence the behavior of cancer cells. The term “cytokine”, as usedherein, refers to small secreted proteins (e.g. IL-1, IL-2, IL-4, IL-5,and IL-6, IFNg, IL-3, IL-7 and GM-CSF) which mediate and regulateimmunity, inflammation, and hematopoiesis. It is shown herein that theantibodies of the prior art are unable to trigger apoptotic cell deathin the absence of stroma cells or stroma-derived cytokines. By contrast,the anti-CD38 antibodies of the invention display under the sameconditions potent apoptotic activities.

In another aspect, the antibodies of the invention are capable ofbinding the CD38 protein with a k_(D) of 3×10⁻⁹ M or lower.

The term “CD38” as used herein refers to a type II transmembraneprotein, comprising, for example, an amino acid sequence as in Genbankaccession number NP_001766. A “CD38⁺ cell” is a cell expressing the CD38protein. Preferably, the CD38⁺ cell is a mammalian cell.

In one embodiment of this invention, the CD38⁺ cell is a malignant cell.In another embodiment, the CD38⁺ cell is a B cell. In a preferredembodiment, the CD38⁺ cell is a tumor cell derived from a hemapoieticmalignancy. In a more preferred embodiment, the CD38⁺ cell is a lymphomacell, a leukemia cell, or a multiple myeloma cell. In a furtherpreferred embodiment, the CD38⁺ cell is a NHL, BL, MM, B-CLL, ALL, TCL,AML, HCL, HL, or CML cell.

Thus, in one embodiment, this invention provides anti-CD38 antibodiescapable of killing at least 24% of Daudi lymphoma cells in the absenceof stroma cells or stroma-derived cytokines. In another embodiment, theanti-CD38 antibodies of the invention are capable of killing at least 7%of Ramos lymphoma cells in the absence of stroma cells or stroma-derivedcytokines. In another embodiment, the anti-CD38 antibodies of theinvention are capable of killing at least 11% of MOLP-8 multiple myelomacells in the absence of stroma cells or stroma-derived cytokines. Inanother embodiment, the anti-CD38 antibodies of the invention arecapable of killing at least 36% of SU-DHL-8 lymphoma cells in theabsence of stroma cells or stroma-derived cytokines. In anotherembodiment, the anti-CD38 antibodies of the invention are capable ofkilling at least 27% of NU-DUL-1 lymphoma cells in the absence of stromacells or stroma-derived cytokines. In another embodiment, the anti-CD38antibodies of the invention are capable of killing at least 62% ofDND-41 leukemia cells in the absence of stroma cells or stroma-derivedcytokines. In another embodiment, the anti-CD38 antibodies of theinvention are capable of killing at least 9% of JVM-13 leukemia cells inthe absence of stroma cells or stroma-derived cytokines. In anotherembodiment, the anti-CD38 antibodies of the invention are capable ofkilling at least 4% of HC-1 leukemia cells in the absence of stromacells or stroma-derived cytokines.

Antibodies

The term “antibody” is used herein in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies) of any isotype such as IgG, IgM, IgA, IgD andIgE, polyclonal antibodies, multispecific antibodies, chimericantibodies, and antibody fragments. An antibody reactive with a specificantigen can be generated by recombinant methods such as selection oflibraries of recombinant antibodies in phage or similar vectors, or byimmunizing an animal with the antigen or an antigen-encoding nucleicacid.

A typical IgG antibody is comprised of two identical heavy chains andtwo identical light chains that are joined by disulfide bonds. Eachheavy and light chain contains a constant region and a variable region.Each variable region contains three segments called“complementarity-determining regions” (“CDRs”) or “hypervariableregions”, which are primarily responsible for binding an epitope of anantigen. They are usually referred to as CDR1, CDR2, and CDR3, numberedsequentially from the N-terminus. The more highly conserved portions ofthe variable regions are called the “framework regions”.

As used herein, “V_(H)” or “VH” refers to the variable region of animmunoglobulin heavy chain of an antibody, including the heavy chain ofan Fv, scFv, dsFv, Fab, Fab′ or F(ab′)2 fragment. Reference to “V_(L)”or “VL” refers to the variable region of the immunoglobulin light chainof an antibody, including the light chain of an Fv, scFv, dsFv, Fab,Fab′ or F(ab′)2 fragment.

A “polyclonal antibody” is an antibody which was produced among or inthe presence of one or more other, non-identical antibodies. In general,polyclonal antibodies are produced from a B-lymphocyte in the presenceof several other B-lymphocytes producing non-identical antibodies.Usually, polyclonal antibodies are obtained directly from an immunizedanimal.

A “monoclonal antibody”, as used herein, is an antibody obtained from apopulation of substantially homogeneous antibodies, i.e. the antibodiesforming this population are essentially identical except for possiblenaturally occurring mutations which might be present in minor amounts.These antibodies are directed against a single epitope and are thereforehighly specific.

An “epitope” is the site on the antigen to which an antibody binds. Ifthe antigen is a polymer, such as a protein or polysaccharide, theepitope can be formed by contiguous residues or by non-contiguousresidues brought into close proximity by the folding of an antigenicpolymer. In proteins, epitopes formed by contiguous amino acids aretypically retained on exposure to denaturing solvents, whereas epitopesformed by non-contiguous amino acids are typically lost under saidexposure.

As used herein, the term “K_(D)” refers to the dissociation constant ofa particular antibody/antigen interaction.

The present invention proceeds from novel murine anti-CD38 antibodies,herein 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 which arefully characterized with respect to the amino acid sequences of bothlight and heavy chains, the identification of the CDRs, theidentification of surface amino acids, and means for their expression inrecombinant form. The primary amino acid and DNA sequences of antibodies38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 light and heavychains, and of humanized versions, are disclosed herein.

The hybridoma cell lines producing the 38SB13, 38SB18, 38SB19, 38SB30,38SB31, and 38SB39 murine anti-CD38 antibodies have been deposited atthe American Type Culture Collection (10801 University Bld, Manassas,Va., 20110-2209, USA), on Jun. 21, 2006, under the deposit numbersPTA-7667, PTA-7669, PTA-7670, PTA-7666, PTA-7668, and PTA-7671,respectively.

The scope of the present invention is not limited to antibodies andfragments comprising these sequences. Instead, all antibodies andfragments that specifically bind to CD38 and capable of killing CD38⁺cells by apoptosis, ADCC, and/or CDC, fall within the scope of thepresent invention. Thus, antibodies and antibody fragments may differfrom antibody 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 or thehumanized derivatives in the amino acid sequences of their scaffold,CDRs, light chain and heavy chain, and still fall within the scope ofthe present invention.

In one embodiment, this invention provides antibodies or epitope-bindingfragment thereof comprising one or more CDRs having an amino acidsequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 81, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36. In a preferredembodiment, the antibodies of the invention comprise at least one heavychain and at least one light chain, and said heavy chain comprises threesequential CDRs having amino acid sequences selected from the groupconsisting of SEQ ID NOS: 1, 2, 3, 7, 8, 9, 13, 81, 15, 19, 20, 21, 25,26, 27, 31, 32, and 33, and said light chain comprises three sequentialCDRs having amino acid sequences selected from the group consisting ofSEQ ID NOS: 4, 5, 6, 10, 11, 12, 16, 17, 18, 22, 23, 24, 28, 29, 30, 34,35, and 36.

In a more preferred embodiment, the antibodies of the invention comprisethree CDRs having amino acid sequences selected from the group of SEQ IDNOS: 1, 2, 3, 4, 5, and 6. In a further more preferred embodiment, thereis provided a 38SB13 antibody, which comprises at least one heavy chainand at least one light chain, and said heavy chain comprises threesequential CDRs having amino acid sequences consisting of SEQ ID NOS: 1,2, and 3, and said light chain comprises three sequential CDRs havingamino acid sequences consisting of SEQ ID NOS: 4, 5, and 6.

In another more preferred embodiment, the antibodies of the inventioncomprise three CDRs having amino acid sequences selected from the groupof SEQ ID NOS: 7, 8, 9, 10, 11, and 12. In a further more preferredembodiment, there is provided a 38SB18 antibody, which comprises atleast one heavy chain and at least one light chain, and said heavy chaincomprises three sequential CDRs having amino acid sequences consistingof SEQ ID NOS: 7, 8, and 9, and said light chain comprises threesequential CDRs having amino acid sequences consisting of SEQ ID NOS:10, 11, and 12.

In another more preferred embodiment, the antibodies of the inventioncomprise three CDRs having amino acid sequences selected from the groupof SEQ ID NOS: 13, 81, 15, 16, 17, and 18. In a further more preferredembodiment, there is provided a 38SB19 antibody, which comprises atleast one heavy chain and at least one light chain, and said heavy chaincomprises three sequential CDRs having amino acid sequences consistingof SEQ ID NOS: 13, 81, and 15, and said light chain comprises threesequential CDRs having amino acid sequences consisting of SEQ ID NOS:16, 17, and 18.

In another more preferred embodiment, the antibodies of the inventioncomprise three CDRs having amino acid sequences selected from the groupof SEQ ID NOS: 19, 20, 21, 22, 23, 24. In a further more preferredembodiment, there is provided a 38SB30 antibody, which comprises atleast one heavy chain and at least one light chain, and said heavy chaincomprises three sequential CDRs having amino acid sequences consistingof SEQ ID NOS: 19, 20, and 21, and said light chain comprises threesequential CDRs having amino acid sequences consisting of SEQ ID NOS:22, 23, and 24.

In another more preferred embodiment, the antibodies of the inventioncomprise three CDRs having amino acid sequences selected from the groupof SEQ ID NOS: 25, 26, 27, 28, 29, and 30. In a further more preferredembodiment, there is provided a 38SB31 antibody, which comprises atleast one heavy chain and at least one light chain, and said heavy chaincomprises three sequential CDRs having amino acid sequences consistingof SEQ ID NOS: 25, 26, and 27, and said light chain comprises threesequential CDRs having amino acid sequences consisting of SEQ ID NOS:28, 29, and 30.

In another more preferred embodiment, the antibodies of the inventioncomprise three CDRs having amino acid sequences selected from the groupof 31, 32, 33, 34, 35, and 36. In a further more preferred embodiment,there is provided a 38SB39 antibody, which comprises at least one heavychain and at least one light chain, and said heavy chain comprises threesequential CDRs having amino acid sequences consisting of SEQ ID NOS:31, 32, and 33, and said light chain comprises three sequential CDRshaving amino acid sequences consisting of SEQ ID NOS: 34, 35, and 36.

In another embodiment, the anti-CD38 antibodies of the inventioncomprise a V_(L) having an amino acid sequence selected from the groupconsisting of SEQ ID NOS: V_(L) for 38, 40, 42, 44, 46, and 48. In amore preferred embodiment, there is provided a 38SB13 antibodycomprising a V_(L) having an amino acid sequence consisting of SEQ IDNO: 38. In a more preferred embodiment, there is provided a 38SB18antibody comprising a V_(L) having an amino acid sequence consisting ofSEQ ID NO: 40. In a more preferred embodiment, there is provided a38SB19 antibody comprising a V_(L) having an amino acid sequenceconsisting of SEQ ID NO: 42. In a more preferred embodiment, there isprovided a 38SB30 antibody comprising a V_(L) having an amino acidsequence consisting of SEQ ID NO: 44. In a more preferred embodiment,there is provided a 38SB31 antibody comprising a V_(L) having an aminoacid sequence consisting of SEQ ID NO: 46. In a more preferredembodiment, there is provided a 38SB39 antibody comprising a V_(L)having an amino acid sequence consisting of SEQ ID NO: 48.

In another embodiment, the antibodies of the invention comprise a V_(H)having an amino acid sequence selected from the group consisting of SEQID NOS: 50, 52, 54, 56, 58, and 60. In a more preferred embodiment,there is provided a 38SB13 antibody comprising a V_(H) having an aminoacid sequence consisting of SEQ ID NO: 50. In a more preferredembodiment, there is provided a 38SB18 antibody comprising a V_(H)having an amino acid sequence consisting of SEQ ID NO: 52. In a morepreferred embodiment, there is provided a 38SB19 antibody comprising aV_(H) having an amino acid sequence consisting of SEQ ID NO: 54. In amore preferred embodiment, there is provided a 38SB30 antibodycomprising a V_(H) having an amino acid sequence consisting of SEQ IDNO: 56. In a more preferred embodiment, there is provided a 38SB31antibody comprising a V_(H) having an amino acid sequence consisting ofSEQ ID NO: 58. In a more preferred embodiment, there is provided a38SB39 antibody comprising a V_(H) having an amino acid sequenceconsisting of SEQ ID NO: 60.

Chimeric and Humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and38SB39 Antibodies

As used herein, a “chimeric antibody” is an antibody in which theconstant region, or a portion thereof, is altered, replaced, orexchanged, so that the variable region is linked to a constant region ofa different species, or belonging to another antibody class or subclass.“Chimeric antibody” also refers to an antibody in which the variableregion, or a portion thereof, is altered, replaced, or exchanged, sothat the constant region is linked to a variable region of a differentspecies, or belonging to another antibody class or subclass. Methods forproducing chimeric antibodies are known in the art. See, e.g., Morrison,1985, Science, 229: 1202; Oi et al., 1986, BioTechniques, 4: 214;Gillies et al., 1989, J. Immunol. Methods, 125: 191-202; U.S. Pat. Nos.5,807,715; 4,816,567; and 4,816,397, which are incorporated herein byreference in their entireties.

In one embodiment of the invention, chimeric versions of 38SB13, 38SB18,38SB19, 38SB30, 38SB31, and 38SB39 are provided. In particular, saidchimeric versions contain at least one human constant region. In a morepreferred embodiment, this human constant region is the human IgG1/Kappaconstant region.

The term “humanized antibody”, as used herein, refers to a chimericantibody which contain minimal sequence derived from non-humanimmunoglobulin. The goal of humanization is a reduction in theimmunogenicity of a xenogenic antibody, such as a murine antibody, forintroduction into a human, while maintaining the full antigen bindingaffinity and specificity of the antibody. Humanized antibodies, orantibodies adapted for non-rejection by other mammals, may be producedusing several technologies such as resurfacing and CDR grafting. As usedherein, the resurfacing technology uses a combination of molecularmodeling, statistical analysis and mutagenesis to alter the non-CDRsurfaces of antibody variable regions to resemble the surfaces of knownantibodies of the target host. The CDR grafting technology involvessubstituting the complementarity determining regions of, for example, amouse antibody, into a human framework domain, e.g., see WO 92/22653.Humanized chimeric antibodies preferably have constant regions andvariable regions other than the complementarity determining regions(CDRs) derived substantially or exclusively from the corresponding humanantibody regions and CDRs derived substantially or exclusively from amammal other than a human.

Strategies and methods for the resurfacing of antibodies, and othermethods for reducing immunogenicity of antibodies within a differenthost, are disclosed in U.S. Pat. No. 5,639,641, which is herebyincorporated in its entirety by reference. Briefly, in a preferredmethod, (1) position alignments of a pool of antibody heavy and lightchain variable regions is generated to give a set of heavy and lightchain variable region framework surface exposed positions wherein thealignment positions for all variable regions are at least about 98%identical; (2) a set of heavy and light chain variable region frameworksurface exposed amino acid residues is defined for a rodent antibody (orfragment thereof); (3) a set of heavy and light chain variable regionframework surface exposed amino acid residues that is most closelyidentical to the set of rodent surface exposed amino acid residues isidentified; (4) the set of heavy and light chain variable regionframework surface exposed amino acid residues defined in step (2) issubstituted with the set of heavy and light chain variable regionframework surface exposed amino acid residues identified in step (3),except for those amino acid residues that are within 5 Å of any atom ofany residue of the complementarity-determining regions of the rodentantibody; and (5) the humanized rodent antibody having bindingspecificity is produced.

Antibodies can be humanized using a variety of other techniquesincluding CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos.5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0519 596; Padlan E. A., 1991, Molecular Immunology 28(4/5): 489-498;Studnicka G. M. et al., 1994, Protein Engineering, 7(6): 805-814;Roguska M. A. et al., 1994, PNAS, 91: 969-973), and chain shuffling(U.S. Pat. No. 5,565,332). Human antibodies can be made by a variety ofmethods known in the art including phage display methods. See also U.S.Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; andinternational patent application publication numbers WO 98/46645, WO98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO91/10741 (said references incorporated by reference in theirentireties).

The present invention provides humanized antibodies or fragmentsthereof, which recognize CD38 and kill CD38⁺ cells by apoptosis, ADCC,and/or CDC. In a further embodiment, the humanized antibodies orepitope-binding fragments thereof have the ability to kill said CD38⁺cells by all three mechanisms. In yet another further embodiment, thehumanized antibodies or epitope-binding fragments thereof of theinvention are capable of killing said CD38⁺ cells by apoptosis even inthe absence of stroma cells or stroma-derived cytokines.

A preferred embodiment of such a humanized antibody is a humanized38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39 antibody, or anepitope-binding fragment thereof.

In more preferred embodiments, there are provided resurfaced orhumanized versions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and38SB39 antibodies wherein surface-exposed residues of the antibody orits fragments are replaced in both light and heavy chains to moreclosely resemble known human antibody surfaces. The humanized 38SB13,38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies or epitope-bindingfragments thereof of the present invention have improved properties. Forexample, humanized 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39antibodies or epitope-binding fragments thereof specifically recognizethe CD38 protein. More preferably, the humanized 38SB13, 38SB18, 38SB19,38SB30, 38SB31, and 38SB39 antibodies or epitope-binding fragmentsthereof have the additional ability to kill a CD38⁺ cell, by apoptosis,ADCC, and/or CDC.

The humanized versions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31,and 38SB39 antibodies are also fully characterized herein with respectto their respective amino acid sequences of both light and heavy chainvariable regions, the DNA sequences of the genes for the light and heavychain variable regions, the identification of the CDRs, theidentification of their surface amino acids, and disclosure of a meansfor their expression in recombinant form. However, the scope of thepresent invention is not limited to antibodies and fragments comprisingthese sequences. Instead, all antibodies and fragments that specificallybind to CD38 and are capable of killing CD38⁺ cells by apoptosis, ADCCand/or CDC fall within the scope of the present invention. Preferably,such antibodies are capable of killing CD38⁺ cells by all threemechanisms. Thus, antibodies and epitope-binding antibody fragments ofthe present invention may differ from the 38SB13, 38SB18, 38SB19,38SB30, 38SB31, and 38SB39 antibodies or the humanized derivativesthereof, in the amino acid sequences of their scaffold, CDRs, and/orlight chain and heavy chain, and still fall within the scope of thepresent invention.

The CDRs of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39antibodies are identified by modeling and their molecular structureshave been predicted. Again, while the CDRs are important for epitoperecognition, they are not essential to the antibodies and fragments ofthe invention. Accordingly, antibodies and fragments are provided thathave improved properties produced by, for example, affinity maturationof an antibody of the present invention.

The sequences of the heavy chain and light chain variable regions of the38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibodies, and thesequences of their CDRs were not previously known and are set forth inthis application. Such information can be used to produce humanizedversions of the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39antibodies. These humanized anti-CD38 antibodies or their derivativesmay also be used as the cell binding agent of the present invention.

Thus, in one embodiment, this invention provides humanized antibodies orepitope-binding fragment thereof comprising one or more CDRs having anamino acid sequence selected from the group consisting of SEQ ID NOS: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 81, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36. In apreferred embodiment, the humanized antibodies of the invention compriseat least one heavy chain and at least one light chain, and said heavychain comprises three sequential CDRs having amino acid sequencesselected from the group consisting of SEQ ID NOS: 1, 2, 3, 7, 8, 9, 13,81, 15, 19, 20, 21, 25, 26, 27, 31, 32, and 33, and said light chaincomprises three sequential CDRs having amino acid sequences selectedfrom the group consisting of SEQ ID NOS: 4, 5, 6, 10, 11, 12, 16, 17,18, 22, 23, 24, 28, 29, 30, 34, 35, and 36. In a further preferredembodiment, a humanized version of 385613 is provided, which comprisesat least one heavy chain and at least one light chain, wherein saidheavy chain comprises three sequential complementarity-determiningregions having amino acid sequences represented by SEQ ID NOS: 1, 2, and3, and wherein said light chain comprises three sequentialcomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOS: 4, 5, and 6. In another further preferredembodiment, a humanized version of 38SB18 is provided, which comprisesat least one heavy chain and at least one light chain, wherein saidheavy chain comprises three sequential complementarity-determiningregions having amino acid sequences represented by SEQ ID NOS: 7, 8, and9, and wherein said light chain comprises three sequentialcomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOS: 10, 11, and 12. In another further preferredembodiment, a humanized version of 38SB19 is provided, which comprisesat least one heavy chain and at least one light chain, wherein saidheavy chain comprises three sequential complementarity-determiningregions having amino acid sequences represented by SEQ ID NOS: 13, 81,and 15, and wherein said light chain comprises three sequentialcomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOS: 16, 17, and 18. In another further preferredembodiment, a humanized version of 38SB30 is provided, which comprisesat least one heavy chain and at least one light chain, wherein saidheavy chain comprises three sequential complementarity-determiningregions having amino acid sequences represented by SEQ ID NOS: 19, 20,and 21, and wherein said light chain comprises three sequentialcomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOS: 22, 23, and 24. In another further preferredembodiment, a humanized version of 38SB31 is provided, which comprisesat least one heavy chain and at least one light chain, wherein saidheavy chain comprises three sequential complementarity-determiningregions having amino acid sequences represented by SEQ ID NOS: 25, 26,and 27, and wherein said light chain comprises three sequentialcomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOS: 28, 29, and 30. In another further preferredembodiment, a humanized version of 38SB39 is provided, which comprisesat least one heavy chain and at least one light chain, wherein saidheavy chain comprises three sequential complementarity-determiningregions having amino acid sequences represented by SEQ ID NOS: 31, 32,and 33, and wherein said light chain comprises three sequentialcomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOS: 34, 35, and 36.

In one embodiment, this invention provides humanized antibodies orfragments thereof which comprise a V_(H) having an amino acid sequenceselected from the group of SEQ ID NOS: 66 and 72. In a preferredembodiment, a humanized 38SB19 antibody is provided which comprises aV_(H) having an amino acid sequence represented by SEQ ID NO: 66. Inanother preferred embodiment, a humanized 38SB31 antibody is providedwhich comprises a V_(H) having an amino acid sequence represented by SEQID NO: 72.

In another embodiment, this invention provides humanized antibodies orfragments thereof which comprise a V_(L) having an amino acid sequenceselected from the group of SEQ ID NOS: 62, 64, 68, and 70. In apreferred embodiment, a humanized 38SB19 antibody is provided whichcomprises a V_(L) having an amino acid sequence chosen from the group ofSEQ ID NOS: 62 and 64. In another preferred embodiment, a humanized38SB31 antibody is provided which comprises a V_(L) having an amino acidsequence chosen from the group of SEQ ID NOS: 68 and 70.

The humanized 38SB19 antibodies and epitope-binding fragments thereof ofthe present invention can also include substitutions in light and/orheavy chain amino acid residues at one or more positions defined by thegrey residues in Table 1A and 1B which represent the murine surfaceframework residues that have been changed from the original murineresidue to the corresponding framework surface residue in the humanantibody, 28E4. The starred (*) residues in Table 1B correspond to themurine back mutations in the humanized 38SB19 heavy chain variant (SEQID NO:65). The residues for back mutations are proximal to CDR's andwere chosen as described in U.S. Pat. No. 5,639,641 or in analogy to theselection of residues that had in previous humanization efforts resultedin a decrease in antigen binding affinity (Roguska et al., 1996, U.S.patent application publications 2003/0235582 and 2005/0118183).

Likewise, the humanized 38SB13, 38SB18, 38SB30, 38SB31, and 38SB39antibodies and epitope-binding fragments thereof of the presentinvention can also include substitution in light and/or heavy chainamino acid residues.

Polynucleotides, Vectors, and Host Cells

Nucleic acids encoding anti-CD38 antibodies of the invention areprovided. In one embodiment, the nucleic acid molecule encodes a heavyand/or a light chain of an anti-CD38 immunoglobulin. In a preferredembodiment, a single nucleic acid encodes a heavy chain of an anti-CD38immunoglobulin and another nucleic acid molecule encodes the light chainof an anti-CD38 immunoglobulin.

In another aspect of this invention, there are provided polynucleotidesencoding polypeptides having an amino acid sequence selected from thegroup of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 81, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, and 72. In a preferred embodiment, the polynucleotide of theinvention is selected from the group consisting of SEQ ID NOs: 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, and 71. Theinvention is not limited to said polynucleotides per se but alsoincludes all polynucleotides displaying at least 80% identity with saidpolynucleotides.

The invention provides vectors comprising the polynucleotides of theinvention. In one embodiment, the vector contains a polynucleotideencoding a heavy chain of an anti-CD38 immunoglobulin. In anotherembodiment, said polynucleotide encodes the light chain of an anti-CD38immunoglobulin. The invention also provides vectors comprisingpolynucleotide molecules encoding, fusion proteins, modified antibodies,antibody fragments, and probes thereof.

In order to express the heavy and/or light chain of the anti-CD38antibodies of the invention, the polynucleotides encoding said heavyand/or light chains are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalsequences. Expression vectors include plasmids, YACs, cosmids,retrovirus, EBV-derived episomes, and all the other vectors that theskilled man will know to be convenient for ensuring the expression ofsaid heavy and/or light chains. The skilled man will realize that thepolynucleotides encoding the heavy and the light chains can be clonedinto different vectors or in the same vector. In a preferred embodiment,said polynucleotides are cloned in the same vector.

Polynucleotides of the invention and vectors comprising these moleculescan be used for the transformation of a suitable mammalian host cell.Transformation can be by any known method for introducingpolynucleotides into a cell host. Such methods are well known of the manskilled in the art and include dextran-mediated transformation, calciumphosphate precipitation, polybrene-mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide intoliposomes, biolistic injection and direct microinjection of DNA intonuclei.

Antibody Fragments

The antibodies of the present invention include both the full lengthantibodies discussed above, as well as epitope-binding fragmentsthereof. As used herein, “antibody fragments” include any portion of anantibody that retains the ability to bind to the epitope recognized bythe full length antibody, generally termed “epitope-binding fragments.”Examples of antibody fragments include, but are not limited to, Fab,Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (dsFv) and fragments comprising either a VL or VHregion. Epitope-binding fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentirety or a portion of the following: hinge region, CH1, CH2, and CH3domains.

Such fragments may contain one or both Fab fragments or the F(ab′)2fragment. Preferably, the antibody fragments contain all six CDRs of thewhole antibody, although fragments containing fewer than all of suchregions, such as three, four or five CDRs, are also functional. Further,the fragments may be or may combine members of any one of the followingimmunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclassesthereof.

Fab and F(ab′)2 fragments may be produced by proteolytic cleavage, usingenzymes such as papain (Fab fragments) or pepsin (F(ab′)2 fragments).

The “single-chain FVs” (“scFvs”) fragments are epitope-binding fragmentsthat contain at least one fragment of an antibody heavy chain variableregion (V_(H)) linked to at least one fragment of an antibody lightchain variable region (V_(L)). The linker may be a short, flexiblepeptide selected to assure that the proper three-dimensional folding ofthe V_(L) and V_(H) regions occurs once they are linked so as tomaintain the target molecule binding-specificity of the whole antibodyfrom which the single-chain antibody fragment is derived. The carboxylterminus of the V_(L) or V_(H) sequence may be covalently linked by alinker to the amino acid terminus of a complementary V_(L) or V_(H)sequence.

Single-chain antibody fragments of the present invention contain aminoacid sequences having at least one of the variable or complementaritydetermining regions (CDRs) of the whole antibodies described in thisspecification, but lack some or all of the constant domains of thoseantibodies. These constant domains are not necessary for antigenbinding, but constitute a major portion of the structure of wholeantibodies. Single-chain antibody fragments may therefore overcome someof the problems associated with the use of antibodies containing a partor all of a constant domain. For example, single-chain antibodyfragments tend to be free of undesired interactions between biologicalmolecules and the heavy-chain constant region, or other unwantedbiological activity. Additionally, single-chain antibody fragments areconsiderably smaller than whole antibodies and may therefore havegreater capillary permeability than whole antibodies, allowingsingle-chain antibody fragments to localize and bind to targetantigen-binding sites more efficiently. Also, antibody fragments can beproduced on a relatively large scale in prokaryotic cells, thusfacilitating their production. Furthermore, the relatively small size ofsingle-chain antibody fragments makes them less likely to provoke animmune response in a recipient than whole antibodies.

Single-chain antibody fragments may be generated by molecular cloning,antibody phage display library or similar techniques well known to theskilled artisan. These proteins may be produced, for example, ineukaryotic cells or prokaryotic cells, including bacteria. Theepitope-binding fragments of the present invention can also be generatedusing various phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In particular, such phage can be utilized to display epitope-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an epitope-binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen bound or captured to a solidsurface or bead. Phage used in these methods are typically filamentousphage including fd and M13 binding domains expressed from phage withFab, Fv or disulfide-stabilized Fv antibody domains recombinantly fusedto either the phage gene III or gene VIII protein.

Examples of phage display methods that can be used to make theepitope-binding fragments of the present invention include thosedisclosed in Brinkman et al., 1995, J. Immunol. Methods, 182: 41-50;Ames et al., 1995, J. Immunol. Methods, 184: 177-186; Kettleborough etal., 1994, Eur. J. Immunol., 24: 952-958; Persic et al., 1997, Gene,187: 9-18; Burton et al., 1994, Advances in Immunology, 57: 191-280;WO/1992/001047; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and5,969,108; each of which is incorporated herein by reference in itsentirety.

After phage selection, the regions of the phage encoding the fragmentscan be isolated and used to generate the epitope-binding fragmentsthrough expression in a chosen host, including mammalian cells, insectcells, plant cells, yeast, and bacteria, using recombinant DNAtechnology, e.g., as described in detail below. For example, techniquesto recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in WO92/22324; Mullinax et al., 1992, BioTechniques, 12(6): 864-869; Sawai etal., 1995, AJRI, 34: 26-34; and Better et al., 1988, Science,240:1041-1043; said references incorporated by reference in theirentireties. Examples of techniques which can be used to producesingle-chain Fvs and antibodies include those described in U.S. Pat.Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods inEnzymology, 203: 46-88; Shu et al., 1993, PNAS, 90: 7995-7999; Skerra etal., 1988, Science, 240:1038-1040.

Functional Equivalents

Also included within the scope of the invention are functionalequivalents of the anti-CD38 antibody and the humanized anti-CD38receptor antibody. The term “functional equivalents” includes antibodieswith homologous sequences, chimeric antibodies, artificial antibodiesand modified antibodies, for example, wherein each functional equivalentis defined by its ability to bind to the CD38 protein. The skilledartisan will understand that there is an overlap in the group ofmolecules termed “antibody fragments” and the group termed “functionalequivalents.” Methods of producing functional equivalents are known tothe person skilled in the art and are disclosed, for example, in WO93/21319, EP 239,400; WO 89/09622; EP 338,745; and EP 332,424, which areincorporated in their respective entireties by reference.

Antibodies with homologous sequences are those antibodies with aminoacid sequences that have sequence homology with amino acid sequence ofan anti-CD38 antibody and a humanized anti-CD38 antibody of the presentinvention. Preferably homology is with the amino acid sequence of thevariable regions of the anti-CD38 antibody and humanized anti-CD38antibody of the present invention. “Sequence homology” as applied to anamino acid sequence herein is defined as a sequence with at least about90%, 91%, 92%, 93%, or 94% sequence homology, and more preferably atleast about 95%, 96%, 97%, 98%, or 99% sequence homology to anotheramino acid sequence, as determined, for example, by the FASTA searchmethod in accordance with Pearson and Lipman, 1988, Proc. Natl. Acad.Sci. USA, 85: 2444-2448.

Artificial antibodies include scFv fragments, diabodies, triabodies,tetrabodies and mru (see reviews by Winter, G. and Milstein, C., 1991,Nature, 349: 293-299; Hudson, P. J., 1999, Current Opinion inImmunology, 11: 548-557), each of which has antigen-binding ability. Inthe single chain Fv fragment (scFv), the V_(H) and VL domains of anantibody are linked by a flexible peptide. Typically, this linkerpeptide is about 15 amino acid residues long. If the linker is muchsmaller, for example 5 amino acids, diabodies are formed, which arebivalent scFv dimers. If the linker is reduced to less than three aminoacid residues, trimeric and tetrameric structures are formed that arecalled triabodies and tetrabodies. The smallest binding unit of anantibody is a CDR, typically the CDR2 of the heavy chain which hassufficient specific recognition and binding that it can be usedseparately. Such a fragment is called a molecular recognition unit ormru. Several such mrus can be linked together with short linkerpeptides, therefore forming an artificial binding protein with higheravidity than a single mru.

The functional equivalents of the present application also includemodified antibodies, e.g., antibodies modified by the covalentattachment of any type of molecule to the antibody. For example,modified antibodies include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Thecovalent attachment does not prevent the antibody from generating ananti-idiotypic response. These modifications may be carried out by knowntechniques, including, but not limited to, specific chemical cleavage,acetylation, formylation, metabolic synthesis of tunicamycin, etc.Additionally, the modified antibodies may contain one or morenon-classical amino acids.

Functional equivalents may be produced by interchanging different CDRson different chains within different frameworks. Thus, for example,different classes of antibody are possible for a given set of CDRs bysubstitution of different heavy chains, whereby, for example, IgG1-4,IgM, IgA1-2, IgD, IgE antibody types and isotypes may be produced.Similarly, artificial antibodies within the scope of the invention maybe produced by embedding a given set of CDRs within an entirelysynthetic framework.

Functional equivalents may be readily produced by mutation, deletionand/or insertion within the variable and/or constant region sequencesthat flank a particular set of CDRs, using a wide variety of methodsknown in the art. The antibody fragments and functional equivalents ofthe present invention encompass those molecules with a detectable degreeof binding to CD38, when compared to the 38SB13, 38SB18, 38SB19, 38SB30,38SB31, or 38SB39 antibody. A detectable degree of binding includes allvalues in the range of at least 10-100%, preferably at least 50%, 60% or70%, more preferably at least 75%, 80%, 85%, 90%, 95% or 99% of thebinding ability of the murine 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or38SB39 antibody to CD38.

Improved Antibodies

The CDRs are of primary importance for epitope recognition and antibodybinding. However, changes may be made to the residues that comprise theCDRs without interfering with the ability of the antibody to recognizeand bind its cognate epitope. For example, changes that do not affectepitope recognition, yet increase the binding affinity of the antibodyfor the epitope may be made.

Thus, also included in the scope of the present invention are improvedversions of both the murine and humanized antibodies, which alsospecifically recognize and bind CD38, preferably with increasedaffinity.

Several studies have surveyed the effects of introducing one or moreamino acid changes at various positions in the sequence of an antibody,based on the knowledge of the primary antibody sequence, on itsproperties such as binding and level of expression (Yang, W. P. et al.,1995, J. Mol. Biol., 254: 392-403; Rader, C. et al., 1998, Proc. Natl.Acad. Sci. USA, 95: 8910-8915; Vaughan, T. J. et al., 1998, NatureBiotechnology, 16: 535-539).

In these studies, equivalents of the primary antibody have beengenerated by changing the sequences of the heavy and light chain genesin the CDR1, CDR2, CDR3, or framework regions, using methods such asoligonucleotide-mediated site-directed mutagenesis, cassettemutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of E.coli (Vaughan, T. J. et al., 1998, Nature Biotechnology, 16: 535-539;Adey, N. B. et al., 1996, Chapter 16, pp. 277-291, in “Phage Display ofPeptides and Proteins”, Eds. Kay, B. K. et al., Academic Press). Thesemethods of changing the sequence of the primary antibody have resultedin improved affinities of the secondary antibodies (Gram, H. et al.,1992, Proc. Natl. Acad. Sci. USA, 89: 3576-3580; Boder, E. T. et al.,2000, Proc. Natl. Acad. Sci. USA, 97: 10701-10705; Davies, J. andRiechmann, L., 1996, Immunotechnolgy, 2: 169-179; Thompson, J. et al.,1996, J. Mol. Biol., 256: 77-88; Short, M. K. et al., 2002, J. Biol.Chem., 277: 16365-16370; Furukawa, K. et al., 2001, J. Biol. Chem., 276:27622-27628).

By a similar directed strategy of changing one or more amino acidresidues of the antibody, the antibody sequences described in thisinvention can be used to develop anti-CD38 antibodies with improvedfunctions, including improved affinity for CD38.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, and (4) conferor modify other physico-chemical or functional properties of suchanalogs. Analogs can include various muteins of a sequence other thanthe naturally-occurring peptide sequence. For example, single ormultiple amino acid substitutions (preferably conservative amino acidsubstitutions) may be made in the naturally-occurring sequence(preferably in the portion of the polypeptide outside the domain (s)forming intermolecular contacts. A conservative amino acid substitutionshould not substantially change the structural characteristics of theparent sequence (e.g., a replacement amino acid should not tend to breaka helix that occurs in the parent sequence, or disrupt other types ofsecondary structure that characterizes the parent sequence). Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed in Proteins, Structures and Molecular Principles (Creighton,Ed., W. H. Freeman and Company, New York (1984)); Introduction toProtein Structure (C. Branden and J. Tooze, eds., Garland Publishing,New York, N.Y. (1991)); and Thornton et al., 1991, Nature, 354: 105,which are each incorporated herein by reference.

Improved antibodies also include those antibodies having improvedcharacteristics that are prepared by the standard techniques of animalimmunization, hybridoma formation and selection for antibodies withspecific characteristics.

Improved antibodies according to the invention include in particularantibodies with enhanced functional properties. Of special interest arethose antibodies with enhanced ability to mediate cellular cytotoxiceffector functions such as ADCC. Such antibodies may be obtained bymaking single or multiple substitutions in the constant framework of theantibody, thus altering its interaction with the Fc receptors. Methodsfor designing such mutants can be found for example in Lazar et al.(2006, Proc. Natl. Acad. Sci. U.S.A. 103(11): 4005-4010) and Okazaki etal. (2004, J. Mol. Biol. 336(5):1239-49). See also WO 03/074679, WO2004/029207, WO 2004/099249, WO2006/047350, WO 2006/019447, WO2006/105338, WO 2007/041635. It is also possible to use cell linesspecifically engineered for production of improved antibodies. Inparticular, these lines have altered regulation of the glycosylationpathway, resulting in antibodies which are poorly fucosylated or eventotally defucosylated. Such cell lines and methods for engineering themare disclosed in e.g. Shinkawa et al. (2003, J. Biol. Chem. 278(5):3466-3473), Ferrara et al. (2006, J. Biol. Chem. 281(8): 5032-5036;2006, Biotechnol. Bioeng. 93(5): 851-61), EP 1331266, EP 1498490, EP1498491, EP 1676910, EP 1792987, and WO 99/54342.

The present invention also includes cytotoxic conjugates. Thesecytotoxic conjugates comprise two primary components, a cell-bindingagent and a cytotoxic agent.

As used herein, the term “cell binding agent” refers to an agent thatspecifically recognizes and binds the CD38 proteins on the cell surface.In one embodiment, the cell binding agent specifically recognizes CD38such that it allows the conjugates to act in a targeted fashion withlittle side-effects resulting from non-specific binding.

In another embodiment, the cell binding agent of the present inventionalso specifically recognizes the CD38 protein so that the conjugateswill be in contact with the target cell for a sufficient period of timeto allow the cytotoxic drug portion of the conjugate to act on the cell,and/or to allow the conjugates sufficient time in which to beinternalized by the cell.

In a preferred embodiment, the cytotoxic conjugates comprise ananti-CD38 antibody as the cell binding agent, more preferably the murine38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39 anti-CD38 monoclonalantibody. In another preferred embodiment, the cell binding agent is achimeric version of said anti-CD38 antibody. In a more preferredembodiment, the cytotoxic conjugate comprises a humanized 38SB13,38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 antibody or anepitope-binding fragment thereof. The 38SB13, 38SB18, 38SB19, 38SB30,38SB31, and 38SB39 antibody is able to specifically recognize CD38, anddirects the cytotoxic agent to an abnormal cell or a tissue, such ascancer cells, in a targeted fashion.

The second component of the cytotoxic conjugates of the presentinvention is a cytotoxic agent. The term “cytotoxic agent” as usedherein refers to a substance that reduces or blocks the function, orgrowth, of cells and/or causes destruction of cells.

In preferred embodiments, the cytotoxic agent is a small drug, aprodrug, a taxoid, a maytansinoid such as DM1 or DM4, a tomaymycinderivative, a leptomycin derivative, CC-1065 or a CC-1065 analog. Inpreferred embodiments, the cell binding agents of the present inventionare covalently attached, directly or via a cleavable or non-cleavablelinker, to the cytotoxic agent.

The cell binding agents, cytotoxic agents, and linkers are discussed inmore detail below.

Cell Binding Agents

The effectiveness of the compounds of the present invention astherapeutic agents depends on the careful selection of an appropriatecell binding agent. Cell binding agents may be of any kind presentlyknown, or that become known, and includes peptides and non-peptides. Thecell binding agent may be any compound that can bind a cell, either in aspecific or non-specific manner. Generally, these can be antibodies(especially monoclonal antibodies), lymphokines, hormones, growthfactors, vitamins, nutrient-transport molecules (such as transferrin),or any other cell binding molecule or substance.

More specific examples of cell binding agents that can be used include:

-   -   a) polyclonal antibodies;    -   b) monoclonal antibodies;    -   c) fragments of antibodies such as Fab, Fab′, and F(ab′)2, Fv        (Parham, 1983, J. Immunol., 131: 2895-2902; Spring et al.,        1974, J. Immunol., 113: 470-478; Nisonoff et al., 1960, Arch.        Biochem. Biophys., 89: 230-244);

In particular, an anti-CD38 monoclonal antibody selected from 38SB13,38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 can be used as a cell bindingagent according to the present invention. Likewise, said cell bindingagent can be a chimeric version of one of the 38SB13, 38SB18, 38SB19,38SB30, 38SB31, and 38SB39 monoclonal antibodies. Preferably, ahumanized anti-CD38 antibody is used as the cell binding agent of thepresent invention. More preferably the humanized anti-CD38 antibody isselected from humanized or resurfaced 38SB13, 38SB18, 38SB19, 38SB30,38SB31, and 38SB39 antibodies.

Cytotoxic Agents

In another embodiment, the humanized antibody or an epitope-bindingfragment thereof can be conjugated to a drug, such as a maytansinoid, toform a prodrug having specific cytotoxicity towards antigen-expressingcells by targeting the drug to the CD38 protein. Cytotoxic conjugatescomprising such antibodies and a small, highly toxic drug (e.g.,maytansinoids, taxanes, tomaymycin derivatives, leptomycin derivatives,and CC-1065 analogs) can be used as a therapeutic for treatment oftumors, such as lymphoma, leukemia, and multiple myeloma.

The cytotoxic agent used in the cytotoxic conjugate of the presentinvention may be any compound that results in the death of a cell, orinduces cell death, or in some manner decreases cell viability.Preferred cytotoxic agents include, for example, maytansinoids andmaytansinoid analogs, taxoids, tomaymycin derivatives, leptomycinderivatives, CC-1065 and CC-1065 analogs, dolastatin and dolastatinanalogs, defined below. These cytotoxic agents are conjugated to theantibodies, antibodies fragments, functional equivalents, improvedantibodies and their analogs as disclosed herein

The cytotoxic conjugates may be prepared by in vitro methods. In orderto link a drug or prodrug to the antibody, a linking group is used.Suitable linking groups are well known in the art and include disulfidegroups, thioether groups, acid labile groups, photolabile groups,peptidase labile groups and esterase labile groups. Preferred linkinggroups are disulfide groups and thioether groups. For example,conjugates can be constructed using a disulfide exchange reaction or byforming a thioether bond between the antibody and the drug or prodrug.

Maytansinoids

Among the cytotoxic agents that may be used in the present invention toform a cytotoxic conjugate, are maytansinoids and maytansinoid analogs.Examples of suitable maytansinoids include maytansinol and maytansinolanalogs. Maytansinoids are drugs that inhibit microtubule formation andthat are highly toxic to mammalian cells.

Examples of suitable maytansinol analogues include those having amodified aromatic ring and those having modifications at otherpositions. Such suitable maytansinoids are disclosed in U.S. Pat. Nos.4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929;4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348;4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

Specific examples of suitable analogues of maytansinol having a modifiedaromatic ring include:

(1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH reductionof ansamytocin P2);(2) C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and(3) C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No.4,294,757) (prepared by acylation using acyl chlorides).

Specific examples of suitable analogues of maytansinol havingmodifications of other positions include:

(1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction ofmaytansinol with H2S or P2S5);(2) C-14-alkoxymethyl (demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);(3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia);(4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by theconversion of maytansinol by Streptomyces);(5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudiflora);(6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (preparedby the demethylation of maytansinol by Streptomyces); and(7) 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titaniumtrichloride/LAH reduction of maytansinol).

In a preferred embodiment, the cytotoxic conjugates of the presentinvention utilize the thiol-containing maytansinoid (DM1), formallytermed N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine, as thecytotoxic agent. DM1 is represented by the following structural formula(I):

In another preferred embodiment, the cytotoxic conjugates of the presentinvention utilize the thiol-containing maytansinoidN2′-deacetyl-N-2′(4-methyl-4-mercapto-1-oxopentyl)-maytansine as thecytotoxic agent. DM4 is represented by the following structural formula(II):

In further embodiments of the invention, other maytansines, includingthiol and disulfide-containing maytansinoids bearing a mono or di-alkylsubstitution on the carbon atom bearing the sulfur atom, may be used.These include a maytansinoid having, at C-3, C-14 hydroxymethyl, C-15hydroxy, or C-20 desmethyl, an acylated amino acid side chain with anacyl group bearing a hindered sulfhydryl group, wherein the carbon atomof the acyl group bearing the thiol functionality has one or twosubstituents, said substituents being CH₃, C₂H₅, linear or branchedalkyl or alkenyl having from 1 to 10 carbon atoms, cyclic alkyl oralkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, orheterocyclic aromatic or heterocycloalkyl radical, and further whereinone of the substituents can be H, and wherein the acyl group has alinear chain length of at least three carbon atoms between the carbonylfunctionality and the sulfur atom.

Such additional maytansines include compounds represented by formula(III):

wherein:Y′ represents(CR₇R₈)_(l)(CR₉=CR₁₀)_(p)(C≡C)_(q)A_(r)(CR₅R₆)_(m)D_(u)(CR₁₁═CR₁₂)_(r)(C≡C)_(s)B_(t)(CR₃R₄)_(n)CR₁R₂SZ,wherein:

-   -   R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or        alkenyl having from 1 to 10 carbon atoms, branched or cyclic        alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,        substituted phenyl or heterocyclic aromatic or heterocycloalkyl        radical, and in addition R₂ can be H;    -   A, B, D are cycloalkyl or cycloalkenyl having 3-10 carbon atoms,        simple or substituted aryl or heterocyclic aromatic or        heterocycloalkyl radical;        R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₁, and R₁₂ are each independently        H, CH₃, C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon        atoms, branched or cyclic alkyl or alkenyl having from 3 to 10        carbon atoms, phenyl, substituted phenyl or heterocyclic        aromatic or heterocycloalkyl radical;    -   l, m, n, o, p, q, r, s, and t are each independently 0 or an        integer of from 1 to 5, provided that at least two of l, m, n,        o, p, q, r, s and t are not zero at any one time; and    -   Z is H, SR or —COR, wherein R is linear alkyl or alkenyl having        from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl        having from 3 to 10 carbon atoms, or simple or substituted aryl        or heterocyclic aromatic or heterocycloalkyl radical.

Preferred embodiments of formula (III) include compounds of formula(III) wherein:

R₁ is H, R₂ is methyl and Z is H.R₁ and R₂ are methyl and Z is H.R₁ is H, R₂ is methyl, and Z is —SCH₃.R₁ and R₂ are methyl, and Z is —SCH₃.

Such additional maytansines also include compounds represented byformula (IV-L), (IV-D), or (IV-D,L):

wherein:Y represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂SZ,wherein:

-   -   R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or        alkenyl having from 1 to 10 carbon atoms, branched or cyclic        alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,        substituted phenyl, or heterocyclic aromatic or heterocycloalkyl        radical, and in addition R₂ can be H;    -   R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, CH₃, C₂H₅,        linear alkyl or alkenyl having from 1 to 10 carbon atoms,        branched or cyclic alkyl or alkenyl having from 3 to 10 carbon        atoms, phenyl, substituted phenyl, or heterocyclic aromatic or        heterocycloalkyl radical;    -   l, m and n are each independently an integer of from 1 to 5, and        in addition n can be 0;    -   Z is H, SR or —COR wherein R is linear or branched alkyl or        alkenyl having from 1 to 10 carbon atoms, cyclic alkyl or        alkenyl having from 3 to 10 carbon atoms, or simple or        substituted aryl or heterocyclic aromatic or heterocycloalkyl        radical; and    -   May represents a maytansinoid which bears the side chain at C-3,        C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl.

Preferred embodiments of formulas (IV-L), (IV-D) and (IV-D,L) includecompounds of formulas (IV-L), (IV-D) and (IV-D,L) wherein:

R₁ is H, R₂ is methyl, R₅, R₆, R₇, and R₈ are each H, l and m are each1, n is 0, and Z is H.R₁ and R₂ are methyl, R₅, R₆, R₇, R₈ are each H, l and m are 1, n is 0,and Z is H.R₁ is H, R₂ is methyl, R₅, R₆, R₇, R₈ are each H, l and m are each 1, nis 0, and Z is —SCH₃.R₁ and R₂ are methyl, R₅, R₆, R₇, R₈ are each H, l and m are 1, n is 0,and Z is —SCH₃.

Preferably the cytotoxic agent is represented by formula (IV-L).

Such additional maytansines also include compounds represented byformula (V):

wherein:Y represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂SZ,wherein:

-   -   R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or        alkenyl having from 1 to 10 carbon atoms, branched or cyclic        alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,        substituted phenyl or heterocyclic aromatic or heterocycloalkyl        radical, and in addition R₂ can be H;    -   R₃, R₄, R₅, R₆, R₇, and R₈ are each independently H, CH₃, C₂H₅,        linear alkyl or alkenyl having from 1 to 10 carbon atoms,        branched or cyclic alkyl or alkenyl having from 3 to 10 carbon        atoms, phenyl, substituted phenyl, or heterocyclic aromatic or        heterocycloalkyl radical;    -   l, m and n are each independently an integer of from 1 to 5, and        in addition n can be 0; and    -   Z is H, SR or —COR, wherein R is linear alkyl or alkenyl having        from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl        having from 3 to 10 carbon atoms, or simple or substituted aryl        or heterocyclic aromatic or heterocycloalkyl radical.

Preferred embodiments of formula (V) include compounds of formula (V)wherein:

R₁ is H, R₂ is methyl, R₅, R₆, R₇, and R₈ are each H; l and m are each1; n is 0; and Z is H.R₁ and R₂ are methyl, R₅, R₆, R₇, and R₈ are each H, l and m are 1; n is0; and Z is H.R₁ is H, R₂ is methyl, R₅, R₆, R₇, and R₈ are each H, l and m are each1, n is 0, and Z is —SCH₃.R₁ and R₂ are methyl, R₅, R₆, R₇, and R₃ are each H, l and m are 1, n is0, and Z is —SCH₃.

Such additional maytansines further include compounds represented byformula (VI-L), (VI-D), or (VI-D,L):

wherein:Y₂ represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂SZ₂,wherein:

-   -   R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or        alkenyl having from 1 to 10 carbon atoms, branched or cyclic        alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,        substituted phenyl or heterocyclic aromatic or heterocycloalkyl        radical, and in addition R₂ can be H;    -   R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, CH₃, C₂H₅,        linear cyclic alkyl or alkenyl having from 1 to 10 carbon atoms,        branched or cyclic alkyl or alkenyl having from 3 to 10 carbon        atoms, phenyl, substituted phenyl or heterocyclic aromatic or        heterocycloalkyl radical;    -   l, m and n are each independently an integer of from 1 to 5, and        in addition n can be 0;    -   Z₂ is SR or COR, wherein R is linear alkyl or alkenyl having        from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl        having from 3 to 10 carbon atoms, or simple or substituted aryl        or heterocyclic aromatic or heterocycloalkyl radical; and    -   May is a maytansinoid.

Such additional maytansines also include compounds represented byformula (VII):

wherein:Y₂′ represents(CR₇R₈)_(l)(CR₉═CR₁₀)_(p)(C≡C)_(q)A_(r)(CR₅R₆)_(m)D_(u)(CR₁₁═CR₁₂)_(r)(C≡C)_(s)B_(t)(CR₃R₄)_(n)CR₁R₂SZ₂,wherein:

-   -   R₁ and R₂ are each independently CH₃, C₂H₅, linear branched or        alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic alkyl        or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted        phenyl or heterocyclic aromatic or heterocycloalkyl radical, and        in addition R₂ can be H;    -   A, B, and D each independently is cycloalkyl or cycloalkenyl        having 3 to 10 carbon atoms, simple or substituted aryl, or        heterocyclic aromatic or heterocycloalkyl radical;    -   R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₁, and R₁₂ are each independently        H, CH₃, C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon        atoms, branched or cyclic alkyl or alkenyl having from 3 to 10        carbon atoms, phenyl, substituted phenyl or heterocyclic        aromatic or heterocycloalkyl radical;    -   l, m, n, o, p, q, r, s, and t are each independently 0 or an        integer of from 1 to 5, provided that at least two of l, m, n,        o, p, q, r, s and t are not zero at any one time; and        Z₂ is SR or —COR, wherein R is linear alkyl or alkenyl having        from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl        having from 3-10 carbon atoms, or simple or substituted aryl or        heterocyclic aromatic or heterocycloalkyl radical.

Preferred embodiments of formula (VII) include compounds of formula(VII) wherein: R₁ is H and R₂ is methyl.

The above-mentioned maytansinoids can be conjugated to anti-CD38antibody 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39 or ahomologue or fragment thereof, wherein the antibody is linked to themaytansinoid using the thiol or disulfide functionality that is presenton the acyl group of an acylated amino acid side chain found at C-3,C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl of the maytansinoid,and wherein the acyl group of the acylated amino acid side chain has itsthiol or disulfide functionality located at a carbon atom that has oneor two substituents, said substituents being CH₃, C₂H₅, linear alkyl oralkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl oralkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl orheterocyclic aromatic or heterocycloalkyl radical, and in addition oneof the substituents can be H, and wherein the acyl group has a linearchain length of at least three carbon atoms between the carbonylfunctionality and the sulfur atom.

A preferred conjugate of the present invention is the one that comprisesthe anti-anti-CD38 antibody 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or38SB39 or a homologue or fragment thereof, conjugated to a maytansinoidof formula (VIII):

wherein:Y₁′ represents(CR₇R₈)_(l)(CR₉═CR₁₀)_(p)(C≡C)_(q)A_(r)(CR₅R₆)_(m)D_(u)(CR₁₁═CR₁₂)_(r)(C≡C)_(s)B_(t)(CR₃R₄)_(n)CR₁R₂S—,wherein:A, B, and D, each independently is cycloalkyl or cycloalkenyl having3-10 carbon atoms, simple or substituted aryl, or heterocyclic aromaticor heterocycloalkyl radical;R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₁, and R₁₂ are each independently H, CH₃,C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branchedor cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical;andl, m, n, o, p, q, r, s, and t are each independently 0 or an integer offrom 1 to 5, provided that at least two of I, m, n, o, p, q, r, s and tare non-not zero at any one time.

Preferably, R₁ is H and R₂ is methyl or R₁ and R₂ are methyl.

An even more preferred conjugate of the present invention is the onethat comprises the anti-CD38 antibody 38SB13, 38SB18, 38SB19, 38SB30,38SB31, or 38SB39 or a homologue or fragment thereof, conjugated to amaytansinoid of formula (IX-L), (IX-D), or (IX-D,L):

wherein:Y₁ represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂S—,wherein:R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or alkenylhaving from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenylhaving from 3 to 10 carbon atoms, phenyl, substituted phenyl,heterocyclic aromatic or heterocycloalkyl radical, and in addition R₂can be H;R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, CH₃, C₂H₅, linearalkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclicalkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substitutedphenyl or heterocyclic aromatic or heterocycloalkyl radical;l, m and n are each independently an integer of from 1 to 5, and inaddition n can be 0; andMay represents a maytansinol which bears the side chain at C-3, C-14hydroxymethyl, C-15 hydroxy or C-20 desmethyl.

Preferred embodiments of formulas (IX-L), (IX-D) and (IX-D,L) includecompounds of formulas (IX-L), (IX-D) and (IX-D,L) wherein:

R₁ is H and R₂ is methyl or R₁ and R₂ are methyl,

-   -   R₁ is H, R₂ is methyl, R₅, R₆, R₇ and R₈ are each H; l and m are        each 1; n is 0,    -   R₁ and R₂ are methyl; R₅, R₆, R₇ and R₈ are each H; l and m are        1; n is 0.

Preferably the cytotoxic agent is represented by formula (IX-L).

An further preferred conjugate of the present invention is the one thatcomprises the anti-CD38 antibody 38SB13, 38SB18, 38SB19, 38SB30, 38SB31,or 38SB39 or a homologue or fragment thereof, conjugated to amaytansinoid of formula (X):

wherein the substituents are as defined for formula (IX) above.

Especially preferred are any of the above-described compounds, whereinR₁ is H, R₂ is methyl, R₅, R₆, R₇ and R₈ are each H, l and m are each 1,and n is 0.

Further especially preferred are any of the above-described compounds,wherein R₁ and R₂ are methyl, R₅, R₆, R₇, R₈ are each H, l and m are 1,and n is 0

Further, the L-aminoacyl stereoisomer is preferred.

Each of the maytansinoids taught in pending U.S. patent application Ser.No. 10/849,136, filed May 20, 2004, may also be used in the cytotoxicconjugate of the present invention. The entire disclosure of U.S. patentapplication Ser. No. 10/849,136 is incorporated herein by reference.

Disulfide-Containing Linking Groups

In order to link the maytansinoid to a cell binding agent, such as the38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39 antibody, themaytansinoid comprises a linking moiety. The linking moiety contains achemical bond that allows for the release of fully active maytansinoidsat a particular site. Suitable chemical bonds are well known in the artand include disulfide bonds, acid labile bonds, photolabile bonds,peptidase labile bonds and esterase labile bonds. Preferred aredisulfide bonds.

The linking moiety also comprises a reactive chemical group. In apreferred embodiment, the reactive chemical group can be covalentlybound to the maytansinoid via a disulfide bond linking moiety.

Particularly preferred reactive chemical groups are N-succinimidylesters and N-sulfosuccinimidyl esters.

Particularly preferred maytansinoids comprising a linking moiety thatcontains a reactive chemical group are C-3 esters of maytansinol and itsanalogs where the linking moiety contains a disulfide bond and thechemical reactive group comprises a N-succinimidyl orN-sulfosuccinimidyl ester.

Many positions on maytansinoids can serve as the position to chemicallylink the linking moiety. For example, the C-3 position having a hydroxylgroup, the C-14 position modified with hydroxymethyl, the C-15 positionmodified with hydroxy and the C-20 position having a hydroxy group areall expected to be useful. However the C-3 position is preferred and theC-3 position of maytansinol is especially preferred.

While the synthesis of esters of maytansinol having a linking moiety isdescribed in terms of disulfide bond-containing linking moieties, one ofskill in the art will understand that linking moieties with otherchemical bonds (as described above) can also be used with the presentinvention, as can other maytansinoids. Specific examples of otherchemical bonds include acid labile bonds, photolabile bonds, peptidaselabile bonds and esterase labile bonds. The disclosure of U.S. Pat. No.5,208,020, incorporated herein, teaches the production of maytansinoidsbearing such bonds.

The synthesis of maytansinoids and maytansinoid derivatives having adisulfide moiety that bears a reactive group is described in U.S. Pat.Nos. 6,441,163 and 6,333,410, and U.S. application Ser. No. 10/161,651,each of which is herein incorporated by reference.

The reactive group-containing maytansinoids, such as DM1, are reactedwith an antibody, such as the 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or38SB39 antibody, to produce cytotoxic conjugates. These conjugates maybe purified by HPLC or by gel-filtration.

Several excellent schemes for producing such antibody-maytansinoidconjugates are provided in U.S. Pat. No. 6,333,410, and U.S. applicationSer. Nos. 09/867,598, 10/161,651 and 10/024,290, each of which isincorporated herein in its entirety.

In general, a solution of an antibody in aqueous buffer may be incubatedwith a molar excess of maytansinoids having a disulfide moiety thatbears a reactive group. The reaction mixture can be quenched by additionof excess amine (such as ethanolamine, taurine, etc.). Themaytansinoid-antibody conjugate may then be purified by gel-filtration.

The number of maytansinoid molecules bound per antibody molecule can bedetermined by measuring spectrophotometrically the ratio of theabsorbance at 252 nm and 280 nm. An average of 1-10 maytansinoidmolecules/antibody molecule is preferred.

Conjugates of antibodies with maytansinoid drugs can be evaluated fortheir ability to suppress proliferation of various unwanted cell linesin vitro. For example, cell lines such as the human lymphoma cell lineDaudi, the human lymphoma cell line Ramos, the human multiple myelomacell line MOLP-8, and the human T acute lymphocytic leukemia line MOLT-4can easily be used for the assessment of cytotoxicity of thesecompounds. Cells to be evaluated can be exposed to the compounds for 24hours and the surviving fractions of cells measured in direct assays byknown methods. IC50 values can then be calculated from the results ofthe assays.

Peg-Containing Linking Groups

Maytansinoids may also be linked to cell binding agents using PEGlinking groups, as set forth in U.S. application Ser. No. 10/024,290.These PEG linking groups are soluble both in water and in non-aqueoussolvents, and can be used to join one or more cytotoxic agents to a cellbinding agent. Exemplary PEG linking groups include hetero-bifunctionalPEG linkers that bind to cytotoxic agents and cell binding agents atopposite ends of the linkers through a functional sulfhydryl ordisulfide group at one end, and an active ester at the other end.

As a general example of the synthesis of a cytotoxic conjugate using aPEG linking group, reference is again made to U.S. application Ser. No.10/024,290 for specific details. Synthesis begins with the reaction ofone or more cytotoxic agents bearing a reactive PEG moiety with acell-binding agent, resulting in displacement of the terminal activeester of each reactive PEG moiety by an amino acid residue of the cellbinding agent, to yield a cytotoxic conjugate comprising one or morecytotoxic agents covalently bonded to a cell binding agent through a PEGlinking group.

Taxanes

The cytotoxic agent used in the cytotoxic conjugates according to thepresent invention may also be a taxane or derivative thereof.

Taxanes are a family of compounds that includes paclitaxel (Taxol), acytotoxic natural product, and docetaxel (Taxotere), a semi-syntheticderivative, two compounds that are widely used in the treatment ofcancer. Taxanes are mitotic spindle poisons that inhibit thedepolymerization of tubulin, resulting in cell death. While docetaxeland paclitaxel are useful agents in the treatment of cancer, theirantitumor activity is limited because of their non-specific toxicitytowards normal cells. Further, compounds like paclitaxel and docetaxelthemselves are not sufficiently potent to be used in conjugates of cellbinding agents.

A preferred taxane for use in the preparation of cytotoxic conjugates isthe taxane of formula (XI):

Methods for synthesizing taxanes that may be used in the cytotoxicconjugates of the present invention, along with methods for conjugatingthe taxanes to cell binding agents such as antibodies, are described indetail in U.S. Pat. Nos. 5,416,064, 5,475,092, 6,340,701, 6,372,738 and6,436,931, and in U.S. application Ser. Nos. 10/024,290, 10/144,042,10/207,814, 10/210,112 and 10/369,563.

Tomaymycin Derivatives

The cytotoxic according to the present invention may also a tomaymycinderivative. Tomaymycin derivatives are pyrrolo[1,4]benzodiazepines(PBDs), a known class of compounds exerting their biological propertiesby covalently binding to the N2 of guanine in the minor groove of DNA.PBDs include a number of minor groove binders such as anthramycin,neothramycin and DC-81.

Novel tomaymycin derivatives that retain high cytotoxicity and that canbe effectively linked to cell binding agents are described in theInternational Application No. PCT/IB2007/000142, whose content is hereinincorporated by reference. The cell binding agent-tomaymycin derivativecomplexes permit the full measure of the cytotoxic action of thetomaymycin derivatives to be applied in a targeted fashion againstunwanted cells only, therefore avoiding side effects due to damage tonon-targeted healthy cells.

The cytotoxic agent according to the present invention comprises one ormore tomaymycin derivatives, linked to a cell binding agent, such as the38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39 antibody, via alinking group. The linking group is part of a chemical moiety that iscovalently bound to a tomaymycin derivative through conventionalmethods. In a preferred embodiment, the chemical moiety can becovalently bound to the tomaymycin derivative via a disulfide bond.

The tomaymycin derivatives useful in the present invention have theformula (XII) shown below:

wherein

represents an optional single bond;

represents either a single bond or a double bond;provided that when

represents a single bond, U and U′, the same or different, independentlyrepresent H, and W and W′, the same or different, are independentlyselected from the group consisting of OH, an ether such as —OR, an ester(e.g. an acetate), such as —OCOR, a carbonate such as —OCOOR, acarbamate such as —OCONRR′, a cyclic carbamate, such that N10 and C11are a part of the cycle, a urea such as —NRCONRR′, a thiocarbamate suchas —OCSNHR, a cyclic thiocarbamate such that N10 and C11 are a part ofthe cycle, —SH, a sulfide such as —SR, a sulphoxide such as —SOR, asulfone such as —SOOR, a sulphonate such as —SO3-, a sulfonamide such as—NRSOOR, an amine such as —NRR′, optionally cyclic amine such that N10and C11 are a part of the cycle, a hydroxylamine derivative such as—NROR′, an amide such as —NRCOR, an azido such as —N3, a cyano, a halo,a trialkyl or triarylphosphonium, an aminoacid-derived group; PreferablyW and W′ are the same or different and are OH, Ome, Oet, NHCONH₂, SMe;and when

represents a double bond, U and U′ are absent and W and W′ represent H;

-   -   R1, R2, R1′, R2′ are the same or different and independently        chosen from Halide or Alkyl optionally substituted by one or        more Hal, CN, NRR′, CF₃, OR, Aryl, Het, S(O)_(q)R, or R1 and R2        and R1′ and R2′ form together a double bond containing group =B        and =B′ respectively.

Preferably, R1 and R2 and R1′ and R2′ form together a double bondcontaining group =B and =B′ respectively.

-   -   B and B′ are the same or different and independently chosen from        Alkenyl being optionally substituted by one or more Hal, CN,        NRR′, CF₃, OR, Aryl, Het, S(O)_(q)R or B and B′ represent an        oxygen atom.

Preferably, B=B′.

More preferably, B=B′=═CH₂ or ═CH—CH₃,

-   -   X, X′ are the same or different and independently chosen from        one or more —O—, —NR—, —(C═O)—, —S(O)_(q)—.

Preferably, X=X′.

More preferably, X=X′=O.

-   -   A, A′ are the same or different and independently chosen from        Alkyl or Alkenyl optionally containing an oxygen, a nitrogen or        a sulfur atom, each being optionally substituted by one or more        Hal, CN, NRR′, CF₃, OR, S(O)_(q)R, Aryl, Het, Alkyl, Alkenyl.

Preferably, A=A′.

More preferably, A=A′=linear unsubstituted alkyl.

-   -   Y, Y′ are the same or different and independently chosen from H,        OR;

Preferably, Y=Y′.

More preferably, Y=Y′=OAlkyl, more preferably OMethyl.

-   -   T is —NR—, —O—, —S(O)_(q)—, or a 4 to 10-membered aryl,        cycloalkyl, heterocyclic or heteroaryl, each being optionally        substituted by one or more Hal, CN, NRR′, CF₃, R, OR, S(O)_(q)R,        and/or linker(s), or a branched Alkyl, optionally substituted by        one or more Hal, CN, NRR′, CF₃, OR, S(O)_(q)R and/or linker(s),        or a linear Alkyl substituted by one or more Hal, CN, NRR′, CF₃,        OR, S(O)_(q)R and/or linker(s).

Preferably, T is a 4 to 10-membered aryl or heteroaryl, more preferablyphenyl or pyridyl, optionally substituted by one or more linker(s).

Said linker comprises a linking group. Suitable linking groups are wellknown in the art and include thiol, sulfide, disulfide groups, thioethergroups, acid labile groups, photolabile groups, peptidase labile groupsand esterase labile groups. Preferred are disulfide groups and thioethergroups.

When the linking group is a thiol-, sulfide (or so-called thioether —S—)or disulfide (—S—S—)-containing group, the side chain carrying thethiol, the sulfide or disulfide group can be linear or branched,aromatic or heterocyclic. One of ordinary skill in the art can readilyidentify suitable side chains.

Preferably, said linker is of formula:

G-D-(Z)p-S—Z′

whereG is a single or double bond, —O—, —S— or —NR—;D is a single bond or -E-, -E-NR—, -E-NR—F—, -E-O—, -E-O—F—, -E-NR—CO—,-E-NR—CO—F—, -E-CO—, —CO-E-, -E-CO—F, -E-S—, -E-S—F, -E-NR—C—S—,-E-NR—CS—F—;where E and F are the same or different and are independently chosenfrom linear or branched —(OCH2CH2)iAlkyl(OCH2CH2)j-,-Alkyl(OCH2CH2)i-Alkyl-, —(OCH2CH2)i-, —(OCH2CH2)iCycloalkyl(OCH2CH2)j-,—(OCH2CH2)iHeterocyclic(OCH2CH2)j-, —(OCH2CH2)iAryl(OCH2CH2)j-,—(OCH2CH2)iHeteroaryl(OCH2CH2)j-, -Alkyl-(OCH2CH2)iAlkyl(OCH2CH2)j-,-Alkyl-(OCH2CH2)i-, -Alkyl-(OCH2CH2)iCycloalkyl(OCH2CH2)j-,-Alkyl(OCH2CH2)iHeterocyclic(OCH2CH2)j-,-Alkyl-(OCH2CH2)iAryl(OCH2CH2)j-, -Alkyl(OCH2CH2)iHeteroaryl(OCH2CH2)j-,-Cycloalkyl-Alkyl-, -Alkyl-Cycloalkyl-, -Heterocyclic-Alkyl-,-Alkyl-Heterocyclic-, -Alkyl-Aryl-, -Aryl-Alkyl-, -Alkyl-Heteroaryl-,-Heteroaryl-Alkyl-;where i and j, identical or different are integers and independentlychosen from 0, 1 to 2000;Z is linear or branched -Alkyl-;p is 0 or 1;Z′ represents H, a thiol protecting group such as COR, R20 or SR20,wherein R20 represents H, methyl, Alkyl, optionally substitutedCycloalkyl, aryl, heteroaryl or heterocyclic, provided that when Z′ isH, said compound is in equilibrium with the corresponding compoundformed by intramolecular cyclisation resulting from addition of thethiol group —SH on the imine bond —NH═ of one of the PBD moieties.

-   -   n, n′, equal or different are 0 or 1.    -   q is 0, 1 or 2.    -   R, R′ are equal or different and independently chosen from H,        Alkyl, Aryl, each being optionally substituted by Hal, CN, NRR′,        CF3, R, OR, S(O)qR, Aryl, Het;        or their pharmaceutically acceptable salts, hydrates, or        hydrated salts, or the polymorphic crystalline structures of        these compounds or their optical isomers, racemates,        diastereomers or enantiomers.

The compounds of the general formula (XII) having geometrical andstereoisomers are also a part of the invention.

The N-10, C-11 double bond of tomaymycin derivatives of formula (XII) isknown to be readily convertible in a reversible manner to correspondingimine adducts in the presence of water, an alcohol, a thiol, a primaryor secondary amine, urea and other nucleophiles. This process isreversible and can easily regenerate the corresponding tomaymycinderivatives in the presence of a dehydrating agent, in a non-proticorganic solvant, in vacuum or at high temperatures (Z. Tozuka, 1983, J.Antibiotics, 36: 276).

Thus, reversible derivatives of tomaymycin derivatives of generalformula (XIII) can also be used in the present invention:

where A, X, Y, n, T, A′, X′, Y′, n′, R1, R2, R1′, R2′ are defined as informula (XII) and W, W′ are the same or different and are selected fromthe group consisting of OH, an ether such as —OR, an ester (e.g. anacetate), such as —OCOR, —COOR, a carbonate such as —OCOOR, a carbamatesuch as —OCONRR′, a cyclic carbamate, such that N10 and C11 are a partof the cycle, a urea such as —NRCONRR′, a thiocarbamate such as —OCSNHR,a cyclic thiocarbamate such that N10 and C11 are a part of the cycle,—SH, a sulfide such as —SR, a sulphoxide such as —SOR, a sulfone such as—SOOR, a sulphonate such as —SO3-, a sulfonamide such as —NRSOOR, anamine such as —NRR′, optionally cyclic amine such that N10 and C11 are apart of the cycle, a hydroxylamine derivative such as —NROR′, an amidesuch as —NRCOR, —NRCONRR′, an azido such as —N3, a cyano, a halo, atrialkyl or triarylphosphonium, an aminoacid-derived group. Preferably,W and W′ are the same or different and are OH, Ome, Oet, NHCONH2, SMe.

Compounds of formula (XIII) may thus be considered as solvates,including water when the solvent is water; these solvates can beparticularly useful.

In a preferred embodiment, the tomaymycin derivatives of the inventionare selected from the group consisting in:

-   8,8′-[1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-methoxy-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[1,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[1,4-butanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[3-methyl-1,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[2,6-pyridinediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[4-(3-tert-butoxycarbonylaminopropyloxy)-2,6-pyridinediylbis-(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-(3-aminopropyloxy)-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-(N-methyl-3-tert-butoxycarbonylaminopropyl)-1,3-benzenediylbis-(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-{5-[3-(4-methyl-4-methyldisulfanyl-pentanoylamino)propyloxy]-1,3-benzenediylbis(methyleneoxy)}-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8, 8′[5-acetylthiomethyl-1,    3-benzenediylbis(methyleneoxy)]-bis[(S)-2-methylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]bis-{2-[(S)-2-methylene-7-methoxy-5-oxo-1,3,11a-tetra    hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy]-ethyl}-carbamic    acid tert-butyl ester-   8,8′-[3-(2-acetylthioethyl)-1,    5-pentanediylbis(oxy)]-bis[(S)-2-methylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-(N-4-mercapto-4,4-dimethylbutanoyl)amino-1,3-benzenediylbis(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-(N-4-methyldithio-4,4-dimethylbutanoyl)-amino-1,3-benzenediylbis(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-(N-methyl-N-(2-mercapto-2,2-dimethylethyl)amino-1,3-benzenediyl(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[5-(N-methyl-N-(2-methyldithio-2,2-dimethylethyl)amino-1,3-benzenediyl(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(2-(4-mercapto-4-methyl)-pentanamido-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(1-(2-(4-methyl-4-methyldisulfanyl)-pentanamido-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(3-(4-methyl-4-methyldisulfanyl)-pentanamido-propoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(4-(4-methyl-4-methyldisulfanyl)-pentanamido-butoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-propyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(1-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-propylybenzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(1-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(1-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-eth    oxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetra    hydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(1-(2-[methyl-(2-methyl-2-methyldisulfanyl-propyl)-amino]-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,    11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(3-[methyl-(4-methyl-4-methyldisulfanyl-pentanoyl)-amino]-propyl)-pyridin-2,    6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,    11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(4-(3-[methyl-(2-methyl-2-methyldisulfanyl-propyl)-amino]-propyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]-   8,8′-[(1-(4-methyl-4-methyldisulfanyl)-pentanamido)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-di    methoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]    as well as the corresponding mercapto derivatives, or their    pharmaceutically acceptable salts, hydrates, or hydrated salts, or    the polymorphic crystalline structures of these compounds or their    optical isomers, racemates, diastereomers or enantiomers.

Preferred compounds are those of formula:

where X, X′, A, A′, Y, Y′, T, n, n′ are defined as above.

The compounds of formula (XII) may be prepared in a number of ways wellknown to those skilled in the art. The compounds can be synthesized, forexample, by application or adaptation of the methods described below, orvariations thereon as appreciated by the skilled artisan. Theappropriate modifications and substitutions will be readily apparent andwell known or readily obtainable from the scientific literature to thoseskilled in the art. In particular, such methods can be found in R. C.Larock, Comprehensive Organic Transformations, Wiley-VCH Publishers,1999.

Methods for synthesizing the tomaymycin derivatives which may be used inthe invention are described in the International Application No.PCT/IB2007/000142. Compounds of the present invention may be prepared bya variety of synthetic routes. The reagents and starting materials arecommercially available, or readily synthesized by well-known techniquesby one of ordinary skill in the arts (see, for example, WO 00/12508, WO00/12507, WO 2005/040170, WO 2005/085260, FR1516743, M. Mori et al.,1986, Tetrahedron, 42: 3793-3806).

The conjugate molecules of the invention may be formed using anytechniques. The tomaymycin derivatives of the invention may be linked toan antibody or other cell binding agent via an acid labile linker, or bya photolabile linker. The derivatives can be condensed with a peptidehaving a suitable sequence and subsequently linked to a cell bindingagent to produce a peptidase labile linker. The conjugates can beprepared to contain a primary hydroxyl group, which can be succinylatedand linked to a cell binding agent to produce a conjugate that can becleaved by intracellular esterases to liberate free derivative.Preferably, the derivatives are synthesized to contain a free orprotected thiol group, and then one or more disulfide orthiol-containing derivatives are each covalently linked to the cellbinding agent via a disulfide bond or a thioether link.

Numerous methods of conjugation are taught in U.S. Pat. Nos. 5,416,064and 5,475,092. The tomaymycin derivatives can be modified to yield afree amino group and then linked to an antibody or other cell bindingagent via an acid labile linker or a photolabile linker. The tomaymycinderivatives with a free amino or carboxyl group can be condensed with apeptide and subsequently linked to a cell binding agent to produce apeptidase labile linker. The tomaymycin derivatives with a free hydroxylgroup on the linker can be succinylated and linked to a cell bindingagent to produce a conjugate that can be cleaved by intracellularesterases to liberate free drug. Most preferably, the tomaymycinderivatives are treated to create a free or protected thiol group, andthen the disulfide- or thiol containing tomaymycin dimers are linked tothe cell binding agent via disulfide bonds.

Preferably, monoclonal antibody- or cell binding agent-tomaymycinderivative conjugates are those that are joined via a disulfide bond, asdiscussed above, that are capable of delivering tomaymycin derivatives.Such cell binding conjugates are prepared by known methods such as bymodifying monoclonal antibodies with succinimidylpyridyl-dithiopropionate (SPDP) (Carlsson et al., 1978, Biochem. J.,173: 723-737). The resulting thiopyridyl group is then displaced bytreatment with thiol-containing tomaymycin derivatives to producedisulfide linked conjugates. Alternatively, in the case of thearyldithio-tomaymycin derivatives, the formation of the cell bindingconjugate is effected by direct displacement of the aryl-thiol of thetomaymycin derivative by sulfhydryl groups previously introduced intoantibody molecules. Conjugates containing 1 to 10 tomaymycin derivativedrugs linked via a disulfide bridge are readily prepared by eithermethod.

More specifically, a solution of the dithio-nitropyridyl modifiedantibody at a concentration of 2.5 mg/ml in 0.05 M potassium phosphatebuffer, at pH 7.5 containing 2 mM EDTA is treated with thethiol-containing tomaymycin derivative (1.3 molar eq./dithiopyridylgroup). The release of thio-nitropyridine from the modified antibody ismonitored spectrophotometrically at 325 nm and is complete in about 16hours. The antibody-tomaymycin derivative conjugate is purified andfreed of unreacted drug and other low molecular weight material by gelfiltration through a column of Sephadex G-25 or Sephacryl S300. Thenumber of tomaymycin derivative moieties bound per antibody molecule canbe determined by measuring the ratio of the absorbance at 230 nm and 275nm. An average of 1-10 tomaymycin derivative molecules/antibody moleculecan be linked via disulfide bonds by this method.

The effect of conjugation on binding affinity towards theantigen-expressing cells can be determined using the methods previouslydescribed by Liu et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93:8618-8623. Cytotoxicity of the tomaymycin derivatives and their antibodyconjugates to cell lines can be measured by back-extrapolation of cellproliferation curves as described in Goldmacher et al., 1985, J.Immunol., 135: 3648-3651. Cytotoxicity of these compounds to adherentcell lines can be determined by clonogenic assays as described inGoldmacher et al., 1986, J. Cell Biol., 102: 1312-1319.

Leptomycin Derivatives

The cytotoxic according to the present invention may also a leptomycinderivative. According to the present invention, “leptomycin derivatives”refer to members of the leptomycin family as defined in Kalesse et al.(2002, Synthesis 8: 981-1003), and includes: leptomycins, such asleptomycin A and leptomycin B, callystatins, ratjadones such asratjadone A and ratjadone B, anguinomycins such as anguinomycin A, B, C,D, kasusamycins, leptolstatin, leptofuranins, such as leptofuranin A, B,C, D. Derivatives of leptomycin A and B are preferred.

More specifically, the derivatives of the invention are of formula (I):

whereinRa and Ra′ are H or -Alk; preferably Ra is -Alk, preferably methyl andRa′ is H;R17 is alkyl optionally substituted by OR, CN, NRR′, perfluoroalkyl;preferably, R17 is alkyl, more preferably methyl or ethyl;R9 is alkyl optionally substituted by OR, CN, NRR′, perfluoroalkyl;preferably, R9 is alkyl, more preferably methyl;X is —O— or —NR—; preferably, X is —NR—;

Y is —U—, —NR—U—, —O—U—, —NR—CO—U—, —U—NR—CO—, —U—CO—, —CO—U—;

preferably, when X is —O—, Y is —U—, —NR—U—, —U—NR—CO—;where U is chosen from linear or branched -Alk-, -Alk(OCH₂CH₂)_(m)—,—(OCH₂CH₂)_(m)-Alk-, -Alk(OCH₂CH₂)_(m)-Alk-, —(OCH₂CH₂)_(m)—,-Cycloalkyl-, -Heterocyclic-, -Cycloalkyl-Alk-, -Alk-Cycloalkyl-,-Heterocyclic-Alk-, -Alk-Heterocyclic-;where m is an integer chosen from 1 to 2000;preferably, U is linear or branched -Alk-,

Z is -Alk-;

n is 0 or 1; preferably n is 0;T represents H, a thiol protecting group such as Ac, R₁ or SR₁, whereinR₁ represents H, methyl, Alk, Cycloalkyl, optionally substituted aryl orheterocyclic, or T represents

where:Ra, Ra′, R17, R9, X, Y, Z, n are defined as above;preferably, T is H or SR₁, wherein R₁ represents Alk, more preferablymethyl;R, R′ identical or different are H or alkyl;Alk represents a linear or branched alkyl; preferably Alk represents(—(CH_(2-q)(CH₃)_(q))_(p)— where p represents an integer from 1 to 10;and q represents an integer from 0 to 2; preferably, Alk represents—(CH₂)— ou —C(CH₃)₂—.or their pharmaceutically acceptable salts, hydrates, or hydrated salts,or the polymorphic crystalline structures of these compounds or theiroptical isomers, racemates, diastereomers or enantiomers.

Preferred compounds may be chosen from:

-   (2-Methylsulfanyl-ethyl)-amid of    (2E,10E,12E,16Z,18E)-(R)-6-Hydroxy-3,5,    7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic    acid-   Bis-[(2-mercaptoethyl)-amid of    (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,    7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic    acid]-   (2-Mercapto-ethyl)-amid of    (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,    15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,    6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic    acid-   (2-Methyldisulfanyl-ethyl)-amid of    (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,    7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic    acid-   (2-Methyl-2-methyldisulfanyl-propyl)-amid of    (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic    acid-   (2-Mercapto-2-methyl-propyl)-amid of (2E,10E,    12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,    17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoic    acid    or their pharmaceutically acceptable salts, hydrates, or hydrated    salts, or the polymorphic crystalline structures of these compounds    or their optical isomers, racemates, diastereomers or enantiomers.

In order to link the derivative to a cell-binding agent, the derivativemust include a moiety (linking group) that allows the derivatives to belinked to a cell binding agent via a linkage such as a disulfide bond, asulfide (or called herein thioether) bond, an acid-labile group, aphoto-labile group, a peptidase-labile group, or an esterase-labilegroup. The derivatives are prepared so that they contain a moietynecessary to link the leptomycin derivative to a cell binding agent via,for example, a disulfide bond, a thioether bond, an acid-labile group, aphoto-labile group, a peptidase-labile group, or an esterase-labilegroup. In order to further enhance solubility in aqueous solutions, thelinking group can contain a polyethylene glycol spacer. Preferably, asulfide or disulfide linkage is used because the reducing environment ofthe targeted cell results in cleavage of the sulfide or disulfide andrelease of the derivatives with an associated increase in cytotoxicity.

Compounds of the present invention may be prepared by a variety ofsynthetic routes. The reagents and starting materials are commerciallyavailable, or readily synthesized by well-known techniques by one ofordinary skill in the art. Methods for synthesizing leptomycinderivatives that may be used in the cytotoxic conjugates of the presentinvention, along with methods for conjugating said leptomycinderivatives to cell binding agents such as antibodies, are described indetail in European Patent Application No. 06290948.6, whose content isincorporated herein by reference.

Cc-1065 Analogues

The cytotoxic agent used in the cytotoxic conjugates according to thepresent invention may also be CC-1065 or a derivative thereof.

CC-1065 is a potent anti-tumor antibiotic isolated from the culturebroth of Streptomyces zelensis. CC-1065 is about 1000-fold more potentin vitro than are commonly used anti-cancer drugs, such as doxorubicin,methotrexate and vincristine (B. K. Bhuyan et al., 1982, Cancer Res.,42, 3532-3537). CC-1065 and its analogs are disclosed in U.S. Pat. Nos.6,372,738, 6,340,701, 5,846,545 and 5,585,499.

The cytotoxic potency of CC-1065 has been correlated with its alkylatingactivity and its DNA-binding or DNA-intercalating activity. These twoactivities reside in separate parts of the molecule. Thus, thealkylating activity is contained in the cyclopropapyrroloindole (CPI)subunit and the DNA-binding activity resides in the two pyrroloindolesubunits.

Although CC-1065 has certain attractive features as a cytotoxic agent,it has limitations in therapeutic use. Administration of CC-1065 to micecaused a delayed hepatotoxicity leading to mortality on day 50 after asingle intravenous dose of 12.5 μg/kg (V. L. Reynolds et al., 1986, J.Antibiotics, XXIX: 319-334). This has spurred efforts to develop analogsthat do not cause delayed toxicity, and the synthesis of simpler analogsmodeled on CC-1065 has been described (M. A. Warpehoski et al., 1988, J.Med. Chem., 31: 590-603).

In another series of analogs, the CPI moiety was replaced by acyclopropabenzindole (CBI) moiety (D. L. Boger et al., 1990, J. Org.Chem., 55: 5823-5833; D. L. Boger et al., 1991, BioOrg. Med. Chem.Lett., 1: 115-120). These compounds maintain the high in vitro potencyof the parental drug, without causing delayed toxicity in mice. LikeCC-1065, these compounds are alkylating agents that bind to the minorgroove of DNA in a covalent manner to cause cell death. However,clinical evaluation of the most promising analogs, Adozelesin andCarzelesin, has led to disappointing results (B. F. Foster et al., 1996,Investigational New Drugs, 13: 321-326; I. Wolff et al., 1996, Clin.Cancer Res., 2: 1717-1723). These drugs display poor therapeutic effectsbecause of their high systemic toxicity.

The therapeutic efficacy of CC-1065 analogs can be greatly improved bychanging the in vivo distribution through targeted delivery to the tumorsite, resulting in lower toxicity to non-targeted tissues, and thus,lower systemic toxicity. In order to achieve this goal, conjugates ofanalogs and derivatives of CC-1065 with cell-binding agents thatspecifically target tumor cells have been described (U.S. Pat. Nos.5,475,092; 5,585,499; 5,846,545). These conjugates typically displayhigh target-specific cytotoxicity in vitro, and exceptional anti-tumoractivity in human tumor xenograft models in mice (R. V. J. Chari et al.,1995, Cancer Res., 55: 4079-4084).

Recently, prodrugs of CC-1065 analogs with enhanced solubility inaqueous medium have been described (European Patent Application No.06290379.4). In these prodrugs, the phenolic group of the alkylatingportion of the molecule is protected with a functionality that rendersthe drug stable upon storage in acidic aqueous solution, and confersincreased water solubility to the drug compared to an unprotectedanalog. The protecting group is readily cleaved in vivo at physiologicalpH to give the corresponding active drug. In the prodrugs described inEP 06290379.4, the phenolic substituent is protected as a sulfonic acidcontaining phenyl carbamate which possesses a charge at physiologicalpH, and thus has enhanced water solubility. In order to further enhancewater solubility, an optional polyethylene glycol spacer can beintroduced into the linker between the indolyl subunit and the cleavablelinkage such as a disulfide group. The introduction of this spacer doesnot alter the potency of the drug.

Methods for synthesizing CC-1065 analogs that may be used in thecytotoxic conjugates of the present invention, along with methods forconjugating the analogs to cell binding agents such as antibodies, aredescribed in detail in EP 06290379.4 (whose content is incorporatedherein by reference) and U.S. Pat. Nos. 5,475,092, 5,846,545, 5,585,499,6,534,660 and 6,586,618 and in U.S. application Ser. Nos. 10/116,053 and10/265,452.

Other Drugs

Drugs such as methotrexate, daunorubicin, doxorubicin, vincristine,vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin,tubulysin and tubulysin analogs, duocarmycin and duocarmycin analogs,dolastatin and dolastatin analogs are also suitable for the preparationof conjugates of the present invention. The drug molecules can also belinked to the antibody molecules through an intermediary carriermolecule such as serum albumin. Doxarubicin and Danorubicin compounds,as described, for example, in U.S. Ser. No. 09/740,991, may also beuseful cytotoxic agents.

Therapeutic Composition

The invention also relates to a therapeutic composition for thetreatment of a hyperproliferative disorder or inflammatory disease or anautoimmune disease in a mammal which comprises a therapeuticallyeffective amount of a compound of the invention and a pharmaceuticallyacceptable carrier. In one embodiment said pharmaceutical composition isfor the treatment of cancer, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;including squamous cell carcinoma; hematopoietic tumors of lymphoidlineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt'slymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, tetratocarcinoma, neuroblastomaand glioma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xerodermapigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma, and other cancers yet to be determined in which CD38 isexpressed predominantly. In a preferred embodiment, the pharmaceuticalcompositions of the invention are used for the treatment of a cancersuch as non-Hodgkin's lymphoma, Hodgkin's lymphoma, hairy cell leukemia,multiple myeloma, chronic lymphocytic leukemia, chronic myeloidleukemia, acute myeloid leukemia, or acute lymphocytic leukemia, inwhich CD38 is expressed, and other cancers yet to be determined in whichCD38 is expressed predominantly. In another embodiment, thepharmaceutical composition of the invention can be used to treatautoimmune diseases, such as systemic lupus erythematosus, rheumatoidarthritis, multiple sclerosis, Crohn's diasease, ulcerative colitis,gastritis, Hashimoto's thyroiditis, ankylosing spondylitis, hepatitisC-associated cryoglobulinemic vasculitis, chronic focal encephalitis,bullous pemphigoid, hemophilia A, membranoproliferativeglomerulnephritis, Sjogren's syndrome, adult and juveniledermatomyositis, adult polymyositis, chronic urticaria, primary biliarycirrhosis, idiopathic thrombocytopenic purpura, neuromyelitis optica,Graves' dysthyroid disease, bullous pemphigoid, membranoproliferativeglonerulonephritis, Churg-Strauss syndrome, and asthma. In anotherembodiment, said pharmaceutical composition relates to other disorderssuch as, for example, graft rejections, such as renal transplantrejection, liver transplant rejection, lung transplant rejection,cardiac transplant rejection, and bone marrow transplant rejection;graft versus host disease; viral infections, such as mV infection, HIVinfection, AIDS, etc.; and parasite infections, such as giardiasis,amoebiasis, schistosomiasis, and others as determined by one of ordinaryskill in the art.

The instant invention provides pharmaceutical compositions comprising:

-   -   a) an effective amount of an antibody, antibody fragment or        antibody conjugate of the present invention, and;    -   b) a pharmaceutically acceptable carrier, which may be inert or        physiologically active.

As used herein, “pharmaceutically-acceptable carriers” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, and the like that are physiologically compatible. Examples ofsuitable carriers, diluents and/or excipients include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanol,and the like, as well as combination thereof. In many cases, it will bepreferable to include isotonic agents, such as sugars, polyalcohols, orsodium chloride in the composition. In particular, relevant examples ofsuitable carrier include: (1) Dulbecco's phosphate buffered saline,pH˜7.4, containing or not containing about 1 mg/ml to 25 mg/ml humanserum albumin, (2) 0.9% saline (0.9% w/v sodium chloride (NaCl)), and(3) 5% (w/v) dextrose; and may also contain an antioxidant such astryptamine and a stabilizing agent such as Tween 20.

The compositions herein may also contain a further therapeutic agent, asnecessary for the particular disorder being treated. Preferably, theantibody, antibody fragment or antibody conjugate of the presentinvention, and the supplementary active compound will have complementaryactivities, that do not adversely affect each other. In a preferredembodiment, the further therapeutic agent is an antagonist ofepidermal-growth factor (EGF), fibroblast-growth factor (FGF),hepatocyte growth factor (HGF), tissue factor (TF), protein C, proteinS, platelet-derived growth factor (PDGF), heregulin,macrophage-stimulating protein (MSP) or vascular endothelial growthfactor (VEGF), or an antagonist of a receptor for epidermal-growthfactor (EGF), fibroblast-growth factor (FGF), hepatocyte growth factor(HGF), tissue factor (TF), protein C, protein S, platelet-derived growthfactor (PDGF), heregulin, macrophage-stimulating protein (MSP), orvascular endothelial growth factor (VEGF), including HER2 receptor, HER3receptor, c-MET, and other receptor tyrosine kinases. In a preferredembodiment, the further therapeutic agent is an agent targeting clustersof differentiation (CD) antigens, including CD3, CD14, CD19, CD20, CD22,CD25, CD28, CD30, CD33, CD36, CD40, CD44, CD52, CD55, CD59, CD56, CD70,CD79, CD80, CD103, CD134, CD137, CD138, and CD152. In a preferredembodiment, the further therapeutic agent is a chemotherapeutic orimmunomodulatory agent.

The compositions of the invention may be in a variety of forms. Theseinclude for example liquid, semi-solid, and solid dosage forms, but thepreferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions. The preferred mode ofadministration is parenteral (e.g. intravenous, intramuscular,intraperinoneal, subcutaneous). In a preferred embodiment, thecompositions of the invention are administered intravenously as a bolusor by continuous infusion over a period of time. In another preferredembodiment, they are injected by intramuscular, subcutaneous,intra-articular, intrasynovial, intratumoral, peritumoral,intralesional, or perilesional routes, to exert local as well assystemic therapeutic effects.

Sterile compositions for parenteral administration can be prepared byincorporating the antibody, antibody fragment or antibody conjugate ofthe present invention in the required amount in the appropriate solvent,followed by sterilization by microfiltration. As solvent or vehicle,there may be used water, saline, phosphate buffered saline, dextrose,glycerol, ethanol, and the like, as well as combination thereof. In manycases, it will be preferable to include isotonic agents, such as sugars,polyalcohols, or sodium chloride in the composition. These compositionsmay also contain adjuvants, in particular wetting, isotonizing,emulsifying, dispersing and stabilizing agents. Sterile compositions forparenteral administration may also be prepared in the form of sterilesolid compositions which may be dissolved at the time of use in sterilewater or any other injectable sterile medium.

The antibody, antibody fragment or antibody conjugate of the presentinvention may also be orally administered. As solid compositions fororal administration, tablets, pills, powders (gelatine capsules,sachets) or granules may be used. In these compositions, the activeingredient according to the invention is mixed with one or more inertdiluents, such as starch, cellulose, sucrose, lactose or silica, underan argon stream. These compositions may also comprise substances otherthan diluents, for example one or more lubricants such as magnesiumstearate or talc, a coloring, a coating (sugar-coated tablet) or aglaze.

As liquid compositions for oral administration, there may be usedpharmaceutically acceptable solutions, suspensions, emulsions, syrupsand elixirs containing inert diluents such as water, ethanol, glycerol,vegetable oils or paraffin oil. These compositions may comprisesubstances other than diluents, for example wetting, sweetening,thickening, flavoring or stabilizing products.

The doses depend on the desired effect, the duration of the treatmentand the route of administration used; they are generally between 5 mgand 1000 mg per day orally for an adult with unit doses ranging from 1mg to 250 mg of active substance. In general, the doctor will determinethe appropriate dosage depending on the age, weight and any otherfactors specific to the subject to be treated.

Therapeutic Methods of Use

In another embodiment, the present invention provides a method forkilling a CD38⁺ cell by administering to a patient in need thereof anantibody which binds said CD38 and is able to kill said CD38⁺ cell byapoptosis, ADCC, and/or CDC. Any of the type of antibodies, antibodyfragments, or cytotoxic conjugates of the invention, may be usedtherapeutically. The invention thus includes the use of anti-CD38monoclonal antibodies, fragments thereof, or cytotoxic conjugatesthereof as medicaments.

In a preferred embodiment, antibodies, antibody fragments, or cytotoxicconjugates of the invention are used for the treatment of ahyperproliferative disorder or inflammatory disease or autoimmunedisease in a mammal. In a more preferred embodiment, one of thepharmaceutical compositions disclosed above, and which contains anantibody, antibody fragment, or cytotoxic conjugate of the invention, isused for the treatment of a hyperproliferative disorder in a mammal. Inone embodiment, the disorder is a cancer. In particular, the cancer is ametastatic cancer.

Accordingly, the pharmaceutical compositions of the invention are usefulin the treatment or prevention of a variety of cancers, including (butnot limited to) the following: carcinoma, including that of the bladder,breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix,thyroid and skin; including squamous cell carcinoma; hematopoietictumors of lymphoid lineage, including leukemia, acute lymphocyticleukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-celllymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloid lineage,including acute and chronic myelogenous leukemias and promyelocyticleukemia; tumors of mesenchymal origin, including fibrosarcoma andrhabdomyoscarcoma; other tumors, including melanoma, seminoma,tetratocarcinoma, neuroblastoma and glioma; tumors of the central andperipheral nervous system, including astrocytoma, neuroblastoma, glioma,and schwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyoscarama, and osteosarcoma; and other tumors, includingmelanoma, xeroderma pigmentosum, keratoactanthoma, seminoma, thyroidfollicular cancer and teratocarcinoma, and other cancers yet to bedetermined in which CD38 is expressed. Preferrably, the disorder is NHL,BL, MM, B-CLL, ALL, TCL, AML, HCL, HL, or CML, in which CD38 isexpressed, and other cancers yet to be determined in which CD38 isexpressed predominantly. In another embodiment, the pharmaceuticalcomposition of the invention can be used to treat autoimmune diseases,such as systemic lupus erythematosus, rheumatoid arthritis, multiplesclerosis, Crohn's diasease, ulcerative colitis, gastritis, Hashimoto'sthyroiditis, ankylosing spondylitis, hepatitis C-associatedcryoglobulinemic vasculitis, chronic focal encephalitis, bullouspemphigoid, hemophilia A, membranoproliferative glomerulnephritis,Sjogren's syndrome, adult and juvenile dermatomyositis, adultpolymyositis, chronic urticaria, primary biliary cirrhosis, idiopathicthrombocytopenic purpura, neuromyelitis optica, Graves' dysthyroiddisease, bullous pemphigoid, membranoproliferative glonerulonephritis,Churg-Strauss syndrome, and asthma. In another embodiment, saidpharmaceutical composition relates to other disorders such as, forexample, graft rejections, such as renal transplant rejection, livertransplant rejection, lung transplant rejection, cardiac transplantrejection, and bone marrow transplant rejection; graft versus hostdisease; viral infections, such as mV infection, HIV infection, AIDS,etc.; and parasite infections, such as giardiasis, amoebiasis,schistosomiasis, and others as determined by one of ordinary skill inthe art.

Similarly, the present invention provides a method for inhibiting thegrowth of selected cell populations comprising contacting target cells,or tissue containing target cells, with an effective amount of anantibody, antibody fragment or antibody conjugate of the presentinvention, or an antibody, antibody fragment or a therapeutic agentcomprising a cytotoxic conjugate, either alone or in combination withother cytotoxic or therapeutic agents. In a preferred embodiment, thefurther therapeutic agent is an antagonist of epidermal-growth factor(EGF), fibroblast-growth factor (FGF), hepatocyte growth factor (HGF),tissue factor (TF), protein C, protein S, platelet-derived growth factor(PDGF), heregulin, macrophage-stimulating protein (MSP) or vascularendothelial growth factor (VEGF), or an antagonist of a receptor forepidermal-growth factor (EGF), fibroblast-growth factor (FGF),hepatocyte growth factor (HGF), tissue factor (TF), protein C, proteinS, platelet-derived growth factor (PDGF), heregulin,macrophage-stimulating protein (MSP), or vascular endothelial growthfactor (VEGF), including HER2 receptor, HER3 receptor, c-MET, and otherreceptor tyrosine kinases. In a preferred embodiment, the furthertherapeutic agent is an agent targeting clusters of differentiation (CD)antigens, including CD3, CD14, CD19, CD20, CD22, CD25, CD28, CD30, CD33,CD36, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80, CD103,CD134, CD137, CD138, and CD152. In a preferred embodiment, the furthertherapeutic agent is a chemotherapeutic or immunomodulatory agent.

The method for inhibiting the growth of selected cell populations can bepracticed in vitro, in vivo, or ex vivo. As used herein, “inhibitinggrowth” means slowing the growth of a cell, decreasing cell viability,causing the death of a cell, lysing a cell and inducing cell death,whether over a short or long period of time.

Examples of in vitro uses include treatments of autologous bone marrowprior to their transplant into the same patient in order to killdiseased or malignant cells; treatments of bone marrow prior to itstransplantation in order to kill competent T cells and preventgraft-versus-host-disease (GVHD); treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen.

The conditions of non-clinical in vitro use are readily determined byone of ordinary skill in the art.

Examples of clinical ex vivo use are to remove tumor cells or lymphoidcells from bone marrow prior to autologous transplantation in cancertreatment or in treatment of autoimmune disease, or to remove T cellsand other lymphoid cells from autologous or allogeneic bone marrow ortissue prior to transplant in order to prevent graft versus host disease(GVHD). Treatment can be carried out as follows. Bone marrow isharvested from the patient or other individual and then incubated inmedium containing serum to which is added the cytotoxic agent of theinvention. Concentrations range from about 10 μM to 1 pM, for about 30minutes to about 48 hours at about 37° C. The exact conditions ofconcentration and time of incubation, i.e., the dose, are readilydetermined by one of ordinary skill in the art. After incubation thebone marrow cells are washed with medium containing serum and returnedto the patient by i.v. infusion according to known methods. Incircumstances where the patient receives other treatment such as acourse of ablative chemotherapy or total-body irradiation between thetime of harvest of the marrow and reinfusion of the treated cells, thetreated marrow cells are stored frozen in liquid nitrogen using standardmedical equipment.

For clinical in vivo use, the antibody, the epitope-binding antibodyfragment, or the cytotoxic conjugate of the invention will be suppliedas solutions that are tested for sterility and for endotoxin levels.Examples of suitable protocols of cytotoxic conjugate administration areas follows. Conjugates are given weekly for 4 weeks as an i.v. boluseach week. Bolus doses are given in 50 to 100 ml of normal saline towhich 5 to 10 ml of human serum albumin can be added. Dosages will be 10μg to 100 mg per administration, i.v. (range of 100 ng to 1 mg/kg perday). More preferably, dosages will range from 50 μg to 30 mg. Mostpreferably, dosages will range from 1 mg to 20 mg. After four weeks oftreatment, the patient can continue to receive treatment on a weeklybasis. Specific clinical protocols with regard to route ofadministration, excipients, diluents, dosages, times, etc., can bedetermined by one of ordinary skill in the art as the clinical situationwarrants.

Diagnostic

The antibodies or antibody fragments of the invention can also be usedto detect CD38 in a biological sample in vitro or in vivo. In oneembodiment, the anti-CD38 antibodies of the invention are used todetermine the level of CD38 in a tissue or in cells derived from thetissue. In a preferred embodiment, the tissue is a diseased tissue. In apreferred embodiment of the method, the tissue is a tumor or a biopsythereof. In a preferred embodiment of the method, a tissue or a biopsythereof is first excised from a patient, and the levels of CD38 in thetissue or biopsy can then be determined in an immunoassay with theantibodies or antibody fragments of the invention. In another preferredembodiment, the level of CD38 is determined on a sample of a tissue orbiopsy thereof, which can be frozen or fixed. The same method can beused to determine other properties of the CD38 protein, such as its cellsurface levels, or its cellular localization.

The above-described method can be used to diagnose a cancer in a subjectknown to or suspected to have a cancer, wherein the level of CD38measured in said patient is compared with that of a normal referencesubject or standard. Said method can then be used to determine whether atumor expresses CD38, which may suggest that the tumor will respond wellto treatment with the antibodies, antibody fragments or antibodyconjugates of the present invention. Preferrably, the tumor is a NHL,BL, MM, B-CLL, ALL, TCL, AML, HCL, HL, or CML, in which CD38 isexpressed, and other cancers yet to be determined in which CD38 isexpressed predominantly.

The present invention further provides for monoclonal antibodies,humanized antibodies and epitope-binding fragments thereof that arefurther labeled for use in research or diagnostic applications. Inpreferred embodiments, the label is a radiolabel, a fluorophore, achromophore, an imaging agent or a metal ion.

A method for diagnosis is also provided in which said labeled antibodiesor epitope-binding fragments thereof are administered to a subjectsuspected of having a cancer or an inflammatory disease or an autoimmunedisease, and the distribution of the label within the body of thesubject is measured or monitored.

Kit

The present invention also includes kits, e.g., comprising a describedcytotoxic conjugate and instructions for the use of the cytotoxicconjugate for killing of particular cell types. The instructions mayinclude directions for using the cytotoxic conjugates in vitro, in vivoor ex vivo.

Typically, the kit will have a compartment containing the cytotoxicconjugate. The cytotoxic conjugate may be in a lyophilized form, liquidform, or other form amendable to being included in a kit. The kit mayalso contain additional elements needed to practice the method describedon the instructions in the kit, such a sterilized solution forreconstituting a lyophilized powder, additional agents for combiningwith the cytotoxic conjugate prior to administering to a patient, andtools that aid in administering the conjugate to a patient.

EXAMPLES

The invention is now described by reference to the following examples,which are illustrative only, and are not intended to limit the presentinvention.

Example 1 Mouse CD38 Antibodies

300-19 cells, a pre-B cell line derived from a Balb/c mouse (M. G. Rethet al. 1985, Nature, 317: 353-355), stably expressing a high level ofhuman CD38 were used for immunization of Balb/c VAF mice. Mice weresubcutaneously immunized with about 5×10⁶ CD38-expressing 300-19 cellsper mouse every 2-3 weeks by standard immunization protocols used atImmunoGen, Inc. The immunized mice were boosted with another dose ofantigen three days before being sacrificed for hybridoma generation. Thespleen from the mouse was collected according to standard animalprotocols and was ground between two sterile, frosted microscopic slidesto obtain a single cell suspension in RPMI-1640 medium. The spleen cellswere pelleted, washed, and fused with murine myeloma P3X63Ag8.653 cells(J. F. Kearney et al. 1979, J Immunol, 123: 1548-1550) by usingpolyethylene glycol-1500 (Roche 783 641). The fused cells wereresuspended in RPMI-1640 selection medium containinghypoxanthine-aminopterin-thymidine (HAT) (Sigma H-0262) and selected forgrowth in 96-well flat-bottomed culture plates (Corning-Costar 3596, 200μL of cell suspension per well) at 37° C. (5% CO2). After 5 days ofincubation, 100 μL of culture supernatant were removed from each welland replaced with 100 μL of RPMI-1640 medium containinghypoxanthine-thymidine (HT) supplement (Sigma H-0137). Incubation at 37°C. (5% CO2) was continued until hydridoma clones were ready for antibodyscreening. Other techniques of immunization and hybridoma production canalso be used, including those described in J. Langone and H. Vunakis(Eds., Methods in Enzymology, Vol. 121, “Immunochemical Techniques, PartI”; Academic Press, Florida) and E. Harlow and D. Lane (“Antibodies: ALaboratory Manual”; 1988; Cold Spring Harbor Laboratory Press, NewYork).

By fluorescence activated cell sorting (FACS) using a Becton DickinsonFACSCalibur or a FACSArray machine, culture supernatants from thehybridoma were screened (with FITC or PE-conjugated anti-mouse IgGantiserum) for secretion of mouse monoclonal antibodies that bind to theCD38-expressing 300-19 cells, but not to the parental 300-19 cells. Thehybridoma clones that tested positive were subcloned, and the isotype ofeach secreted anti-CD38 antibody was identified using commercialisotyping reagents (Roche 1493027). A total of 29 antibodies that werepositive for CD38 binding were purified by Protein A or G chromatographyusing a standard protocol and then characterized further.

Example 2 Binding Characterization of Anti-CD38 Antibodies

FACS histograms demonstrating the binding of anti-CD38 antibodies,38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 to CD38-expressing300-19 cells and the absence of binding to the parental 300-19 cells areshown in FIG. 1. 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39antibody (10 nM) was incubated for 3 h with either CD38-expressing300-19 cells or the parental 300-19 cells (1-2×10⁵ cells per sample) in100 μL ice-cold RPMI-1640 medium supplemented with 2% normal goat serum.Then, the cells were pelleted, washed, and incubated for 1 h on ice withFITC-conjugated goat anti-mouse IgG-antibody (Jackson Laboratory, 100μL, 6 μg/mL in cold RPMI-1640 medium supplemented with 2% normal goatserum). The cells were pelleted again, washed, resuspended in 200 μL ofPBS containing 1% formaldehyde, and analyzed using a FACSCalibur flowcytometer with CellQuest software (BD Biosciences).

The FACS histograms of CD38-expressing 300-19 cells incubated with38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or 38SB39 showed a strongfluorescence shift, compared to that of the corresponding negativecontrol (cells incubated only with FITC-conjugated, goat anti-mouseIgG-antibody) (FIG. 1). Also, no significant fluorescence shift wasdetected when parental 300-19 cells were incubated with any of theseantibodies. Similar results were obtained when the positive controlanti-CD38 antibody, AT13/5 (Serotec, MCA1019) was used.

A strong fluorescence shift was also observed when Ramos (ATCC CRL 1596)lymphoma cells were incubated with 38SB13, 38SB18, 38SB19, 38SB30,38SB31, or 38SB39 (FIG. 1). The values for the apparent dissociationconstants (KD) of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 forthe binding to Ramos cells were estimated from the FACS analysis curvesshown in FIG. 2, using the non-linear regression method for sigmoidaldose response curves (GraphPad Prizm, version 4, software, San Diego,Calif.). The values are as follows: 0.10 nM, 0.10 nM, 0.12 nM, 0.16 nM,0.11 nM, and 3.03 nM, respectively.

Example 3 Induction of Apoptosis of Ramos and Daudi Lymphoma Cells, by38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 Antibodies.

The anti-CD38 antibodies, 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and38SB39 induced apoptosis of Ramos and Daudi (ATCC CCL-213) lymphoma celllines and the MOLP-8 multiple myeloma cell line (DSMZ ACC 569). Thedegree of apoptosis was measured by FACS analysis after staining withFITC conjugates of Annexin V (Biosource PHN1018) and with TO-PRO-3(Invitrogen T3605). Annexin V binds phosphatidylserine on the outsidebut not on the inside of the cell membrane bilayer of intact cells. Inhealthy, normal cells, phosphatidylserine is expressed on the inside ofthe membrane bilayer, and the transition of phosphatidylserine from theinner to the outer leaflet of the plasma membrane is one of the earliestdetectable signals of apoptosis. Binding of Annexin V is thus a signalfor the induction of apoptosis. TO-PRO-3 is a monomeric cyanine nucleicacid stain that can only penetrate the plasma membrane when the membraneintegrity is breached, as occurs in the later stages of apoptosis.

Exponentially growing cells were plated at about 2×10⁵ cells/mL in24-well plates in RMPI-1640 medium supplemented with 10% fetal bovineserum (FBS), 2 mM L-glutamine, and 50 μg/mL gentamycin (denoted below ascomplete RMPI-1640 medium). Cells were generally grown in completeRMPI-1640 medium, unless stated otherwise. Cells were incubated withanti-CD38 antibodies (10 nM) for 24 h at 37° C. in a humidifiedatmosphere containing 5% CO₂. The cells were then pelleted, washed twicewith 500 μL PBS, resuspended in 100 μL binding buffer (provided in theAnnexin V-FITC kit), containing 5 μL of Annexin V-FITC, and incubatedfor 15 min on ice. Then, 400 μL of binding buffer and TO-PRO-3 (to afinal concentration of 1 μM) was added to the mix, and thecell-associated fluorescence of FITC and TO-PRO-3 was immediatelymeasured by FACS. Four thousand events were collected for each sample.The dot plots for fluorescence of TO-PRO-3 (FL4-H; y-axis) andfluorescence of Annexin V-FITC (FL1-H; x-axis) were generated usingCellQuest software.

The results are shown in FIGS. 3 and 4. FIG. 3 gives an example of sucha dot plot for Daudi cells after a 24-h incubation with variousanti-CD38 antibodies. The average percentages of Annexin V-positivecells (includes both TO-PRO-3 positive and negative cells) fromduplicate samples were determined from these plots and are shown in FIG.4. Unexpectedly, 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39showed strong apoptotic activities. Greater than 30% of Daudi cellsexposed to any of these antibodies were Annexin V-positive, compared toonly about 6% of untreated cells (FIGS. 3 and 4A). 38SB13, 38SB18,38SB19, 38SB30, 38SB31, and 38SB39 showed at least 2.4-fold strongerapoptotic activities (24% after subtraction of the non-treated controlvalue) than prior art murine CD38 antibodies tested at the sameconcentration of 10 nM, (AT13/5, OKT10, IB4, and SUN-4B7, less than 10%Annexin V-positive after subtraction of the non-treated control value)and two other anti-CD38 antibodies generated in our laboratory, (38SB7and 38SB23, not higher than non-treated control, i. e. about 6% AnnexinV-positive) (FIG. 4A). AT13/5 was purchased from Serotec (MCA1019), andOKT10 was produced and purified from hybridoma (ATCC CRL-8022). IB4 andSUN-4B7 was a gift from Prof. F. Malavasi, University of Turin, Italy.Similarly, 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 anti-CD38antibodies displayed at least 3.5-fold stronger pro-apoptotic activityon another lymphoma cell line, Ramos (7% or more Annexin-V-positiveafter subtraction of the non-treated control value) than either priorart murine CD38 antibodies, AT13/5, OKT10, 164, and SUN-4B7, or twoother new anti-CD38 antibodies, 38SB7 and 38SB23 (less than 2% AnnexinV-positive after subtracting the non-treated control value) (FIG. 4B).Finally, 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39 anti-CD38antibodies displayed strong pro-apoptotic activity on the multiplemyeloma cell line MOLP-8 (FIG. 4C). Approximately 50% of MOLP-8 cellstreated with these antibodies were Annexin V-positive, compared to about39% of untreated cells. In contrast, treatment with any of the prior artmurine CD38 antibodies, AT13/5, OKT10, IB4, and SUN-4B7, or two othernew anti-CD38 antibodies, 38SB7 and 38SB23 resulted on no increase inthe portion of apoptotic cells.

Example 4 Cloning and Sequencing of the Light and Heavy Chains ofAnti-CD38 Antibodies 38SB19 Antibody.

RNA Preparation from Hybridoma Cells that Produces the 38SB19 Antibody

Preparations of total RNA were obtained from 5×10⁶ hybridoma cells,which produce 38SB19 antibody, using Qiagen's RNeasy miniprep kit.Briefly, 5×10⁶ cells were pelleted and resuspended in 350 μL RLT buffer(containing 1% β-mercaptoethanol). The suspension was homogenized bypassing it through a 21.5 gauge needle and syringe roughly 10-20 timesor until it was no longer viscous. Ethanol (350 μL of 70% aqueousethanol) was added to the homogenate, which was mixed well. The solutionwas transferred to a spin column, placed in a 2-mL collection tube andspun at >8000×g for 15 seconds. The column was washed twice with 500 μLRPE buffer, then transferred to a fresh tube and eluted with 30 μL RNasefree water and a 1-minute spin. The eluate (30 μL) was placed back onthe column for a second 1-minute elution spin. An aliquot of the 30 μLeluate was diluted with water and used to measure the UV absorption at260 nm for RNA quantitation.

cDNA Preparation with Reverse Transcriptase (RT) Reaction

The variable region 38SB19 antibody cDNA was generated from the totalRNA using Invitrogen's SuperscriptII kit. The kit protocols werefollowed closely, utilizing up to 5 μg of total RNA from the Qianeasymini preps. Briefly, the RNA, 1 μL random primers, and 1 μL dNTP mixwere brought up to 12 μL with RNase free sterile distilled water andincubated at 65° C. for 5 minutes. The mix was then put on ice for atleast 1 minute. Next 4 μL of 5× reaction buffer, 2 μL 0.1 M DTT, and 1μL RNaseOUT were added and the mix was incubated at 25° C. for 2 minutesin an MJ Research thermalcycler. The thermalcylcer was paused so that 1μL of SuperscriptII enzyme could be added and then restarted for anadditional 10 minutes at 25° C. before shifting to 55° C. for 50minutes. The reaction was heat inactivated by heating to 70° C. for 15min and the RNA was removed by adding 1 μL RNase H and incubating at 37°C. for 20 minutes.

Degenerate PCR Reactions

The procedure for the first round degenerate PCR reaction on the cDNAderived from hybridoma cells was based on methods described in Wang etal. (2000) and Co et al. (1992). The primers for this round (Table 2)contain restriction sites to facilitate cloning into the pBluescriptIIplasmids.

The PCR reaction components (Table 3) were mixed on ice in thin walledPCR tubes and then transferred to an MJ research thermalcycler preheatedand paused at 94° C. The reactions were performed using a programderived from Wang et al., 2000 as follows:

Name: Wang45 94° C. 3:00 min 94° C. 0:15 sec 45° C. 1:00 min 72° C. 2:00min Goto 2 29 times 72° C. 6:00 min  4° C. for ever end

The PCR reaction mixtures were then run on a 1% low melt agarose gel,the 300 to 400 bp bands were excised, purified using Zymo DNA minicolumns, and sent to Agencourt biosciences for sequencing. Therespective 5′ and 3′ PCR primers were used as sequencing primers togenerate the 38SB19 variable region cDNAs from both directions.

Cloning the 5′ End Sequence

Since the degenerate primers used to clone the 38SB19 variable regionlight chain and heavy chain cDNA sequences alters the 5′ end sequences,additional sequencing efforts were needed to decipher the completesequences. The preliminary cDNA sequence from the methods describedabove were used to search the NCBI IgBlast site(http://www.ncbi.nlm.nih.gov/igblast/) for the murine germline sequencesfrom which the 38SB19 sequence is derived. PCR primers were designed(Table 3) to anneal to the leader sequence of the murine antibody sothat a new PCR reaction could yield the complete variable region cDNA,unaltered by the PCR primers. The PCR reactions, band purifications, andsequencing were performed as described above.

Peptide Analysis for Sequence Confirmation

The cDNA sequence information for the variable region was combined withthe germline constant region sequence to obtain full length antibodycDNA sequences. The molecular weights of the heavy chain and light chainwere then calculated and compared with the molecular weights obtained byLC/MS analyses of the murine 38SB19 antibody.

Table 4 gives the calculated mass from the cDNA sequences for 38SB19 LCand HC together with the values measured by LC/MS. The molecular weightmeasurements are consistent with the cDNA sequences for both the 38SB19light and heavy chain.

Example 5

Recombinant Expression of hu38SB19 Antibodies

The variable region sequences for hu38SB19 were codon-optimized andsynthesized by Blue Heron Biotechnology. The sequences are flanked byrestriction enzyme sites for cloning in-frame with the respectiveconstant sequences in both single chain and the tandem dual chainmammalian expression plasmids. The light chain variable region is clonedinto EcoRI and BsiWI sites in both the ps38SB19LCZv1.0 and ps38SB19v1.00plasmids (FIGS. 5A and 5C). The heavy chain variable region is clonedinto the HindIII and Apa1 sites in both the ps38SB19HCNv1.0 andps38SB19v1.00 plasmids (FIGS. 5B and 5C). These plasmids can be used toexpress hu38SB19 in either transient or stable transfections inmammalian cells. Similar expression vector constructs were used toproduce other chimeric and humanized antibodies.

Transient transfections to express hu38SB19 in HEK-293T cells wereperformed using CaPO₄ reagents from BD biosciences. The suppliedprotocols were slightly modified for enhanced expression yields.Briefly, 2×10⁶ HEK-293T cells were plated on 10 cm tissue culture platescoated with polyethyleneimine (PEI) 24 h prior to transfection. Thetransfection began by washing the cells with PBS and replacing the mediawith 10 mL DMEM (Invitrogen) with 1% Ultra Low IgG FBS (Hyclone).Solution A (10 μg DNA, 86.8 μL Ca²⁺ solution, and up to 500 μL with H₂O)was added drop wise to Solution B while vortexing. The mixture wasincubated at RT for 1 min and 1 mL of the mixture was added drop wise toeach 10 cm plate. Approximately 16 h post transfection, media wasreplaced with 10 mL fresh DMEM with 1% Ultra Low IgG FBS. Approximately24 hours later 2 mM sodium butyrate was added to each 10 cm plate. Thetransfection was harvested 4 days later.

Supernatant was prepared for Protein A affinity chromatography by theaddition of 1/10 volume of 1 M Tris/HCl buffer, pH 8.0. The pH-adjustedsupernatant was filtered through a 0.22 μm filter membrane and loadedonto a Protein A Sepharose column (HiTrap Protein A HP, 1 mL, AmershamBiosciences) equilibrated with binding buffer (PBS, pH 7.3). AQ-Sepharose precolumn (10 mL) was connected upstream of the Protein Acolumn during sample loading to reduce contamination from cellularmaterial such as DNA. Following sample loading, the precolumn wasremoved and the Protein A column orientation was reversed for wash andelution. The column was washed with binding buffer until a stablebaseline was obtained with no absorbance at 280 nm. Antibody was elutedwith 0.1 M acetic acid buffer containing 0.15 M NaCl, pH 2.8, using aflow rate of 0.5 mL/min. Fractions of approximately 0.25 mL werecollected and neutralized by the addition of 1/10 volume of 1M Tris/HCl,pH 8.0. The peak fraction(s) was dialysed overnight twice against PBSand purified antibody was quantitated by absorbance at OD₂₈₀. Humanizedand chimeric antibodies can also be purified using a Protein G columnwith slightly different procedures.

All the described chimeric and humanized anti-CD38 antibodies wereexpressed and purified in similar procedures as described above.

Example 6 Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)Activities of Chimeric Anti-CD38 Antibodies.

Since some anti-CD38 antibodies have been previously shown to have ADCCand/or CDC activity as chimeric or humanized antibodies with human IgG1constant regions (J. H. Ellis et al. 1995, J Immunol, 155: 925-937; F.K. Stevenson et al. 1991, Blood, 77: 1071-1079; WO 2005/103083), thechimeric versions of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, and 38SB39,consisting of murine variable regions and the human IgG1/IgKappaconstant region, were made and tested for ADCC and/or CDC activities.

Ch38SB13, ch38SB18, ch38SB19, ch38SB30, ch38SB31, and ch38SB39 werefirst tested for ADCC using Ramos cells as target cells and humannatural killer (NK) cells as effector cells. A lactate dehydrogenase(LDH) release assay was used to measure cell lysis (R. L. Shields etal., 2001, J Biol Chem, 276: 6591-6604).

The NK cells were first isolated from human blood (from a normal donor;purchased from Research Blood Components, Inc., Brighton, Mass.) using amodified protocol for NK Isolation Kit II (Miltenyi Biotech). Blood wasdiluted 2-3-fold with Hank's Balanced Salt Solution (HBSS). Twenty fivemL of diluted blood was carefully layered over 25 mL of Ficoll Paque ina 50 mL conical tube and centrifuged at 400 g for 45 min at 19° C. Theperipheral blood mononuclear cells (PBMC) were collected from theinterface, transferred into a new conical 50 mL tube, and washed oncewith HBSS. The PBMC were resuspended in 2 mL of NK-isolation buffer, andthen 500 μL of Biotin-Antibody Cocktail (from the NK-isolation kit,130-091-152, Miltenyi Biotech) were added to the cell suspension. TheBiotin-Antibody Cocktail contains biotinylated antibodies that bind tothe lymphocytes, except for NK cells. The mixture was incubated at 4° C.for 10 min, and then 1.5 mL of NK-isolation buffer (PBS, 0.1% BSA, 1 mMEDTA) and 1 mL of Anti-Biotin Micro Beads was added. The cell-antibodymixture was incubated for another 15 min at 4° C. Next, cells werewashed once with 50 mL of NK-isolation buffer and resuspended in 3 mL ofNK-isolation buffer. Then, MACS LS column (on the MACS separator,Miltenyi Biotech) was pre-washed with 3 mL of NK-isolation Buffer. Thecell suspension was then applied onto the LS column. The effluent(fraction with unlabeled cells) was collected into a new 50-mL conicaltube. The column was washed 3 times with 3 mL of NK-isolation Buffer.The entire effluent was collected into the same tube and washed oncewith 50 mL of NK-isolation Buffer. NK cells were plated into 30 mL ofRPMI-1640 supplemented with 5% fetal bovine serum, 50 μg/mL gentamycin.

Various concentrations of ch38SB13, ch38SB18, ch38SB19, ch38SB30,ch38SB31, and ch38SB39 antibodies in RPMI-1640 medium supplemented with0.1% BSA, 20 mM HEPES, pH 7.4, and 50 μg/mL gentamycin (denoted below asRHBP medium) were aliquoted (50 μL/well) into a round bottom 96-wellplate. The target Ramos cells were resuspended at 10⁶ cells/mL in RHBPmedium and added to each well (100 μL/well) containing antibodydilutions. The plate containing target cells and antibody dilutions wasincubated for 30 min at 37° C. NK cells (50 μL/well) were then added tothe wells containing the target cells typically at a ratio of 1 targetcell to 3-6 NK cells ratio. RHBP medium (50 μL/well) was added to thecontrol wells with NK cells. Also, 20 μL of Triton X-100 solution(RPMI-1640 medium, 10% Triton X-100) was added to the 3 wells containingonly target cells without antibody, to determine the maximum possibleLDH release. The mixtures were incubated at 37° C. for 4 h, thencentrifuged for 10 min at 1200 rpm, and 100 μL of the supernatant wascarefully transferred to a new flat-bottom 96-well plate. LDH reactionmixture (100 μL/well) from Cytotoxicity Detection Kit (Roche 1 644 793)was added to each well and incubated at room temperature for 5-30 min.The optical density of samples was measured at 490 nm (OD₄₉₀). Thepercent specific lysis of each sample was determined by ascribing 100%lysis to the OD₄₉₀ value of Triton X-100-treated samples and 0% lysis tothe OD₄₉₀ value of the untreated control sample containing only targetcells. The samples containing only NK cells gave negligible OD₄₉₀readings.

When tested with Ramos target cells and NK effector cells, chimericanti-CD38 antibodies showed very potent ADCC activities (FIG. 6). TheEC₅₀ values were estimated by a non-linear regression method withsigmoidal dose response curves and found to be as follows: 0.0013 μg/mLfor ch38SB13, 0.0013 μg/mL for ch38SB18, 0.0018 μg/mL for ch38SB19,0.0022 μg/mL for ch38SB30, 0.0012 μg/mL for ch38SB31, 0.1132 μg/mL forch38SB39. Chimeric anti-CD38 antibodies also showed potent ADCC activityon LP-1 (DSMZ ACC 41) multiple myeloma cells (EC₅₀ values: 0.00056 μg/mLfor ch38SB18; 0.00034 μg/mL for ch38SB19; 0.00024 μg/mL for ch38SB31)(FIG. 7A). Ch38SB19 also efficiently killed Daudi lymphoma cells (FIG.7B), NALM-6 B-ALL cells (DSMZ ACC 128) (FIG. 8A), and MOLT-4 T-ALL cells(ATTC CRL-1582) (FIG. 8B) by ADCC, suggesting anti-CD38 antibodies withunusually potent apoptotic activity also have potent ADCC activityagainst various tumor cells derived from various hematopoieticmalignancies. Also, a non-binding IgG1 control antibody (rituximab,BiogenIdec) (FIGS. 7A, 8A, and 8B) or mu38SB19 (FIG. 7B) in the sameexperiment had no significant ADCC activity.

Example 7 CDC Activities of Chimeric Anti-CD38 Antibodies.

The CDC activities of ch38SB13, ch38SB18, ch38SB19, ch38SB30, ch38SB31,and ch38SB39 were measured based on a method modified from (H.Gazzano-Santoro et al. 1997, J. Immunol Methods, 202: 163-171). Humancomplement was lyophilized human complement serum (Sigma-Aldrich S1764)that was reconstituted with sterile purified water as indicated by themanufacturer and then diluted five-fold with RHBP media immediatelybefore the experiment. Target cells suspended at 10⁶ cells/mL in RHBPmedium were aliquoted into wells of a flat-bottom 96-well tissue cultureplate (50 μL/well). Then, 50 μL of various concentrations (from 10 nM to0.001 nM) of the anti-CD38 antibodies in RHBP medium were added (oneantibody per sample), which was followed by 50 μL/well of complementsolution. The plate was then incubated for 2 h at 37° C. in a humidifiedatmosphere containing 5% CO₂, after which time 50 μL of 40% Alamar Bluereagent (Biosource DAL1100) diluted in RHBP (10% final) was added toeach well to measure the viability of the cells. Alamar Blue monitorsthe reducing capacity of the viable cells. The plate was incubated for5-18 h at 37° C. before measuring the fluorescence (in relativefluorescence units, RFU) at 540/590 nm. The percentage of specific cellviability for each sample was determined by first correcting theexperimental values for background fluorescence by subtracting thebackground RFU value (wells with medium only, without any cells) fromthe RFU values for each sample, and then, dividing the corrected RFUvalues by the corrected RFU value of untreated cell samples.

When the CDC activities of the chimeric anti-CD38 antibody samples weretested with Raji-IMG cells using human complement at a final dilution of5%, chimeric anti-CD38 antibodies showed very potent CDC activities(FIG. 9). Raji-IMG are cells derived from Raji cells (ATCC CCL-86) andexpress lower levels of the membrane complement inhibitors CD55 andCD59. The EC₅₀ values were estimated by non-linear regression from thesigmoidal dose response curve shown in FIG. 8. and are as follows: 0.005μg/mL for ch38SB13, 0.0101 μg/mL for ch38SB18, 0.028 μg/mL for ch38SB19,0.020 μg/mL for ch38SB30, 0.010 μg/mL for ch38SB31, and 0.400 μg/mL forch38SB39. Chimeric anti-CD38 antibodies also showed potent CDC activitytowards LP-1 multiple myeloma cells (EC₅₀ value: 0.032 μg/mL forch38SB18; 0.030 μg/mL for ch38SB19; 0.043 μg/mL for ch38SB31), while anon-binding chimeric control IgG1 (rituximab, BiogenIdec) did not haveany CDC activity (FIG. 10). When chimeric CD38 antibodies were tested onDaudi lymphoma cells, different anti-CD38 antibodies differed in theirCDC activities (FIG. 11). While the specific viability of Daudi cellswas less than 15% following their incubation with 1.25 μg/mL of ch38SB19in the presence of complement, there was only a marginal decrease in thespecific viability of these cells following their incubation withch38SB18 or ch38SB39 (1.25 μg/mL or higher concentration) in thepresence of complement (the specific viability was 85% and 91%,respectively). Also, only a modest reduction of specific viability wasobserved when the Daudi cells were incubated with 1.25 μg/mL or higherconcentration of ch38SB13, ch38SB30, and ch38SB31 in the presence ofcomplement (the specific viability was 65%, 45%, and 53%, respectively).

Example 8 Binding Affinity and Apoptotic, ADCC. And CDC Activities ofHumanized Anti-CD38 Antibodies.

The two versions of humanized 38SB19 (hu38SB19 v1.00 and v1.20) and thechimeric 38SB19 showed similar binding affinities when tested with Ramoscells with K_(D) values of 0.23 nM, 0.25 nM, and 0.18 nM, respectively(FIG. 12A). The binding affinities of chimeric and humanized 38SB19antibodies were also compared in a competiton binding assay, where theirability to compete with the binding of biotinylated murine 38SB19antibody is measured. Biotin-labeled murine 38SB19 antibody (3×10⁻¹⁰ M)was mixed with various concentrations of ch38SB19, hu38SB19 v1.00, orhu38SB19 v1.20. The antibody mixture was incubated with Ramos cells, andthe amount of the biotinylated murine 38SB19 bound to the cells wasmeasured with FITC-conjugated streptavidin by FACS analysis. Hu38SB19v1.00, hu38SB19 v1.20, and ch38SB19 competed with the binding ofbiotinylated murine 38SB19 equally well (FIG. 12B), again indicatingthat the binding affinity was unaffected by the humanization. Whench38SB19, hu38SB19 v1.00 and hu38SB19 v1.20 (10⁻⁸ to 10⁻¹¹ M) werecompared for their ability to induce apoptosis of Daudi cells, theyshowed similar apoptotic activities (FIG. 13). Moreover, hu38SB19 v1.00and v1.20 also showed similar ADCC as ch38SB19 in LP-1 cells (FIG. 14)and similar CDC potencies as ch38SB19 in Raji-IMG and LP-1 cells (FIG.15). Hu38SB19 v1.00 also showed similar CDC activity as ch38SB19 in theT-cell acute lymphoblastic leukemia cell line DND-41 (DSMZ 525) (FIG.15). Hu38SB19 v1.00 was further tested for its ability to induceapoptosis in a diverse set of cell lines (FIG. 16). Treatment withhu38SB19 v1.00 (10⁻⁸ M) resulted in a dramatic increase of AnnexinV-positive cells in the B cell lymphoma cell lines SU-DHL-8 (DSMZ ACC573) (from 7% in untreated control to 97% in hu38SB19-treated cells) andNU-DUL-1 (DSMZ ACC 579) (from 10% in untreated control to 37% inhu38SB19-treated cells) and the T-ALL cell line DND-41 (from 7% inuntreated control to 69% in hu38SB19-treated cells). In addition,treatment with hu38SB19 v1.00 (10⁻⁸ M) increased the portion of AnnexinV-positive cells in the B-cell lymphocytic leukemia cell line JVM-13(DSMZ ACC 19) (from 8% in untreated control to 17% in hu38SB19-treatedcells) and in the hairy cell leukemia cell line HC-1 (DSMZ ACC 301)(from 6% in untreated control to 10% in hu38SB19-treated cells).

Similarly, two versions of humanized 38SB31 (hu38SB31 v1.1 and v1.2) andthe chimeric 38SB31 showed similar binding affinities when tested withRamos cells with K_(D) values of 0.13 nM, 0.11 nM, and 0.12 nM,respectively. The binding affinities of chimeric and humanized 38SB31antibodies were also compared in a competition binding assay, asdescribed above and performed equally well. Hu38SB31v1.1 was furthertested for its ability to induce apoptosis in several cell lines. Thehumanized antibody showed similar apoptotic activities as ch38SB31towards Ramos, Daudi, Molp-8 and SU-DHL-8 cells. Moreover, hu38SB31 v1.1also showed similar ADCC and CDC activities as ch38SB31 in these celllines.

Example 9 In Vivo Efficacy of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31,and 38SB39.

In vivo anti-tumor activities of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31,and 38SB39 were investigated in a survival human xenograft tumor modelin immunodeficient mice (female CB.17 SCID) established with Ramoslymphoma cells. Female CB.17 SCID mice were inoculated with 2×10⁶ Ramoscells in 0.1 mL serum-free medium through a lateral tail vein. Sevendays after tumor cell inoculation, mice were randomized into sevengroups based on body weight. There were 10 mice per group, except forthe 38SB31-treated group, which had 6 mice, and the 38SB39-treatedgroup, which had 8 mice. Antibodies were given to mice intravenously ata dose of 40 mg/kg, twice per week, in three successive weeks, startingseven days after cell inoculation. Mice were sacrificed if one or bothhind legs were paralyzed, the loss of body weight was more than 20% fromthe pre-treatment value, or the animal was too sick to reach food andwater. The treatment with 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, or38SB39 significantly extended the survival of mice compared to that ofPBS-treated mice (FIG. 17). The median survival of PBS-treated mice was22 days and that of antibody-treated groups ranged from 28 to 33 days.

In vivo anti-tumor activities of mu38SB19 and hu38SB19 were furtherinvestigated in additional human xenograft tumor models inimmunodeficient mice. For a Daudi lymphoma survival model, SCID micewere inoculated with 5×10⁶ Daudi cells in 0.1 mL serum-free mediumthrough a lateral tail vein. The study was carried out as describedabove. The treatment with either mu38SB19 or hu38SB19 significantlyextended the survival of mice compared to that of PBS-treated mice (FIG.18). The median survival of PBS-treated mice was 22 days, while themedian survival of antibody-treated mice was 47 days.

For a NCI-H929 multiple myeloma tumor model, SCID mice were inoculatedsubcutaneously with 10⁷ cells. When tumors were palpable on day 6, theanimals were randomized into groups of 10 according to body weight andantibody treatment was started. The hu38SB19 antibody or a non-bindingchimeric IgG1 control antibody (rituximab, BiogenIdec) were given tomice intravenously at a dose of 40 mg/kg, twice per week, in threesuccessive weeks. Tumor volume was monitored and animals were sacrificedif tumors reached 2000 mm³ in size or became necrotic. The PBS treatedgroup reached a mean tumor volume of 1000 mmm³ on day 89, the chimericIgG1 control antibody group on day 84 (FIG. 19). Treatment with hu38SB19completely prevented tumor growth in all 10 animals. In contrast, onlytwo animals in the PBS treated group and three animals in the chimericIgG1 control antibody showed tumor regression.

For a MOLP-8 multiple myeloma tumor model, SCID mice were inoculatedsubcutaneously with 10⁷ cells. When tumors were palpable on day 4, theanimals were randomized into groups of 10 according to body weight andantibody treatment was started. The hu38SB19 and mu38SB19 antibodies ora chimeric IgG1 control antibody were given to mice intravenously at adose of 40 mg/kg, twice per week, in three successive weeks. Tumorvolume was monitored and animals were sacrificed if tumors reached 2000mm³ in size or became necrotic. The PBS treated group reached a meantumor volume of 500 mmm³ on day 22, the chimeric IgG1 control antibodygroup on day 23 (FIG. 20). None of the tumors in these groups regressed.In contrast, treatment with hu38SB19 or mu38SB19 led to tumor regressionin 8 of 10 or 6 of 10 animals, respectively.

Tables

TABLE 1A The mu38SB19 light chain framework surface residues andcorresponding residues at the same Kabat position in the human 1.69antibody. The residues that are different and therefore changed in thehu38SB19 antibody are shown. The starred (*) residues are back mutatedto the mu38SB19 residue in one or more hu38SB19 variants. mu38SB19 LightChain Framework Surface Residues And Corresponding Residues In The Human1.69 Antibody Kabat Position mu38SB19 1.69  1 D D  3 V V  5* T A  9 L K10 S F 15 L V 18 P R 40 P P 41 G G 42 Q Q 45 R K 57 G G 60 D D 67 A S 80A A 81 E E 107  K K 108  R R

TABLE 1B The mu38SB19 heavy chain framework surface residues andcorresponding residues at the same Kabat position in the human 1.69antibody. The residues that are different and therefore changed in thehu38SB19 antibody are shown. mu38SB19 Heavy Chain Framework SurfaceResidues And Corresponding Residues in The Human 1.69 Antibody KabatPosition mu38SB19 1.69  1 Q Q  3 Q Q  5 V Q  9 A A 11 V L 13 K R 14 P P19 K K 23 K K 28 T T 41 P P 42 G G 43 Q Q 61 Q Q 62 K K 64 Q K 65 G G 73K K 74 S S   82B S S 84 S S 85 E E 106  Q Q 113  S S

TABLE 2Primers used for the degenerate PCR reactions are based on those in Wang et al., 2000 except HindKL which is based on Co et al.1992. Mixed bases are defined as follows:H = A + T + C, S = g + C, Y = C + T, K = G + T, M = A + C,R = A + g, W = A + T, V = A + C + G. Primer Sequence BamIgG1GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC (SEQ ID NO. 73) IgG2AbamGGAGGATCCCTTGACCAGGCATCCTAGAGTCA (SEQ ID NO. 74) EcoMH1CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC (SEQ ID NO. 75) EcoMH2CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG (SEQ ID NO. 76) SacIMKGGAGCTCGAYATTGTGMTSACMCARWCTMCA (SEQ ID NO. 77) HindKLTATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTT (SEQ ID NO. 78) GGTGC

TABLE 3 The light and heavy chain PCR reaction mixes for cloning of the38SB19 variable region cDNA sequences. Light Chain Reaction Mix HeavyChain Reaction Mix 5 μl 10 X PCR reaction buffer 5 μl 10 X PCR reactionbuffer (Roche) (Roche) 4 μl 10 mM dNTP mix (2.5 mM 4 μl 10 mM dNTP mix(2.5 mM each) each) 2 μl Template (RT reaction) 2 μl Template (RTreaction) 5 μl 10 μM Sac1MK left primer 2.5 μl 10 μM EcoMH1 left primer5 μl 10 μM HindKL right primer 2.5 μl 10 μM EcoMH2 left primer 5 μl 10μM BamIgG1 right primer 5 μl DMSO 5 μl DMSO 0.5 μl Taq Polymerase(Roche) 0.5 μl Taq Polymerase (Roche) 23.5 μl sterile distilled H₂O 23.5μl sterile distilled H₂O 50 μl Total 50 μl Total

TABLE 4 The 5′ end murine leader sequence primers used forthe 38SB19 second round PCR reactions. The 3′ endprimers are identical to those used in the firstround reactions since they prime to therespective constant region sequences. Primer Sequence Light ChainATGGAGTCACAGATTCAGGTC 38SB19 LC Leader (SEQ ID NO. 79) Heavy ChainTTTTGAATTCCAGTAACTTCAGGTGTCCACTC 38-19HCLead1 (SEQ ID NO. 80)

TABLE 5 cDNA calculated and LC/MS measured molecular weights of themurine 38SB19 antibody light and heavy chains. Light Chain Heavy ChaincDNA LC/MS Difference cDNA LC/MS Difference Mu38SB19 23735 23736 1 4880548826 21

1: An antibody or epitope-binding fragment thereof that specificallybinds CD38, characterized in that said antibody or epitope-bindingfragment thereof is capable of killing a CD38⁺ cell by apoptosis,antibody-dependent cell-mediated cytotoxicity (ADCC), andcomplement-dependent cytotoxicity (CDC). 2: An antibody orepitope-binding fragment thereof according to claim 1, characterized inthat said antibody or epitope-binding fragment thereof is capable ofkilling said CD38⁺ cell by apoptosis in the absence of stroma cells orstroma-derived cytokines. 3: An antibody or epitope-binding fragmentthereof according to claim 1, characterized in that said antibody orepitope-binding fragment thereof is a monoclonal antibody. 4: Anantibody or epitope-binding fragment thereof according to claim 1,characterized in that said CD38⁺ cell is a lymphoma cell, a leukemiacell, or a multiple myeloma cell. 5: The antibody or epitope-bindingfragment thereof of claim 4, characterized in that said CD38⁺ cell is anon-Hodgkin's lymphoma (NHL) cell, a Burkitt's lymphoma (BL) cell, amultiple myeloma (MM) cell, a B chronic lymphocytic leukemia (B-CLL)cell, a B and T acute lymphocytic leukemia (ALL) cell, a T cell lymphoma(TCL) cell, an acute myeloid leukemia (AML) cell, a hairy cell leukemia(HCL) cell, a Hodgkin's Lymphoma (HL) cell, or a chronic myeloidleukemia (CML) cell. 6-13. (canceled) 14: An antibody or epitope-bindingfragment thereof according to claim 1, characterized in that saidantibody or epitope-binding fragment thereof binds CD38 with a K_(D) of3×10⁻⁹M or lower. 15-37. (canceled) 38: An antibody or epitope-bindingfragment thereof according to claim 1, characterized in that saidantibody or epitope-binding fragment thereof comprises at least onehuman constant region. 39: An antibody or epitope-binding fragmentthereof according to claim 38, characterized in that said constantregion is the human IgG1/IgKappa constant region. 40: An antibody orepitope-binding fragment thereof according to claim 1, characterized inthat said antibody or epitope-binding fragment thereof is a humanized orresurfaced antibody. 41: A humanized or resurfaced antibody orepitope-binding fragment thereof according to claim 40, characterized inthat said humanized or resurfaced antibody or epitope-binding fragmentthereof comprises at least one heavy chain and at least one light chain,wherein said heavy chain comprises three sequentialcomplementarity-determining regions of SEQ ID NOS: 1, 2, and 3, andwherein said light chain comprises three sequentialcomplementarity-determining regions of SEQ ID NOS: 4, 5, and
 6. 42: Ahumanized or resurfaced antibody or epitope-binding fragment thereofaccording to claim 40, characterized in that said humanized orresurfaced antibody or epitope-binding fragment thereof comprises atleast one heavy chain and at least one light chain, wherein said heavychain comprises three sequential complementarity-determining regions ofSEQ ID NOS: 7, 8, and 9, and wherein said light chain comprises threesequential complementarity-determining regions of SEQ ID NOS: 10, 11,and
 12. 43-45. (canceled) 46: A humanized or resurfaced antibody orepitope-binding fragment thereof according to claim 40, characterized inthat said humanized or resurfaced antibody or epitope-binding fragmentthereof comprises at least one heavy chain and at least one light chain,wherein said heavy chain comprises three sequentialcomplementarity-determining regions of SEQ ID NOS: 19, 20, and 21, andwherein said light chain comprises three sequentialcomplementarity-determining regions SEQ ID NOS: 22, 23, and
 24. 47: Ahumanized or resurfaced antibody or epitope-binding fragment thereofaccording to claim 40, characterized in that said humanized orresurfaced antibody or epitope-binding fragment thereof comprises atleast one heavy chain and at least one light chain, wherein said heavychain comprises three sequential complementarity-determining regions ofSEQ ID NOS: 25, 26, and 27, and wherein said light chain comprises threesequential complementarity-determining regions of SEQ ID NOS: 28, 29,and
 30. 48: A humanized or resurfaced antibody or epitope-bindingfragment thereof according to claim 47, characterized in that saidhumanized or resurfaced antibody or epitope-binding fragment thereofcomprises a heavy chain variable region comprising SEQ ID NO: 72 and alight chain variable region comprising SEQ ID NO: 68 or SEQ ID NO: 70.49. (canceled) 50: A humanized or resurfaced antibody or epitope-bindingfragment thereof according to claim 40, characterized in that saidhumanized or resurfaced antibody or epitope-binding fragment thereofcomprises at least one heavy chain and at least one light chain, whereinsaid heavy chain comprises three sequential complementarity-determiningregions of SEQ ID NOS: 31, 32, and 33, and wherein said light chaincomprises three sequential complementarity-determining regions of SEQ IDNOS: 34, 35, and
 36. 51: An antibody or epitope-binding fragment thereofaccording to claim 1, characterized in that said antibody orepitope-binding fragment thereof is a Fab, Fab′, F(ab′)2 or Fv fragment.52: A conjugate comprising the antibody or epitope-binding fragmentthereof according to claim 1 linked to a cytotoxic agent. 53: Theconjugate of claim 52, characterized in that said cytotoxic agent isselected from the group consisting of a maytansinoid, a small drug, atomaymycin derivative, a leptomycin derivative, a prodrug, a taxoid,CC-1065 and a CC-1065 analog. 54: The conjugate of claim 53,characterized in that said cytotoxic agent is the maytansine DM1 offormula:

55: The conjugate of claim 53, characterized in that said cytotoxicagent is the maytansine DM4 of formula:

56: The conjugate of claim 53, characterized in that said cytotoxicagent is a tomaymycin derivative selected from the group consisting of:8,8′-[1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-methoxy-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[1,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[1,4-butanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[3-methyl-1,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[2,6-pyridinediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[4-(3-tert-butoxycarbonylaminopropyloxy)-2,6-pyridinediylbis-(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-(3-aminopropyloxy)-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-(N-methyl-3-tert-butoxycarbonylaminopropyl)-1,3-benzenediylbis-(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-{5-[3-(4-methyl-4-methyldisulfanyl-pentanoylamino)propyloxy]-1,3-benzenediylbis(methyleneoxy)}-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-acetylthiomethyl-1,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-methylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]bis-{2-[(S)-2-methylene-7-methoxy-5-oxo-1,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy]-ethyl}-carbamicacid tert-butyl ester8,8′-[3-(2-acetylthioethyl)-1,5-pentanediylbis(oxy)]-bis[(S)-2-methylene-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-(N-4-mercapto-4,4-dimethylbutanoyl)amino-1,3-benzenediylbis(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-(N-4-methyldithio-4,4-dimethylbutanoyl)-amino-1,3-benzenediylbis(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-(N-methyl-N-(2-mercapto-2,2-dimethylethyl)amino-1,3-benzenediyl(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3, 11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[5-(N-methyl-N-(2-methyldithio-2,2-dimethylethyl)amino-1,3-benzenediyl(methyleneoxy)]-bis[7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(2-(4-mercapto-4-methyl)-pentanamido-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(1-(2-(4-methyl-4-methyldisulfanyl)-pentanamido-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(3-(4-methyl-4-methyldisulfanyl)-pentanamido-propoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(4-(4-methyl-4-methyldisulfanyl)-pentanamido-butoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-propyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(1-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-propyl)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(1-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(1-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(1-(2-[methyl-(2-methyl-2-methyldisulfanyl-propyl)-amino]-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(3-[methyl-(4-methyl-4-methyldisulfanyl-pentanoyl)-amino]-propyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]8,8′-[(4-(3-[methyl-(2-methyl-2-methyldisulfanyl-propyl)-amino]-propyl)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]and8,8′-[(1-(4-methyl-4-methyldisulfanyl)-pentanamido)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrolo[2,1-c][1,4]benzodiazepin-5-one].57. (canceled) 58: The conjugate of claim 53, characterized in that thecytotoxic agent is a leptomycin derivative selected from the groupconsisting of: (2-Methyl sulfanyl-ethyl)-amid of(2E,10E,12E,16Z,18E)-(R)-6-Hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoicacid (2-methylsulfanyl-ethyl)-amid Bis-[(2-mercaptoethyl)-amid of(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoicacid] (2-Mercapto-ethyl)-amid of(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoicacid (2-Methyldisulfanyl-ethyl)-amid of(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoicacid (2-Methyl-2-methyldisulfanyl-propyl)-amid of(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoicacid and (2-Mercapto-2-methyl-propyl)-amid of(2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo-nonadeca-2,10,12,16,18-pentaenoicacid. 59-60. (canceled) 61: A pharmaceutical composition comprising (a)the antibody or epitope-binding fragment thereof according to claim 1 orthe conjugate according to claim 52, and (b) a pharmaceuticallyacceptable carrier or excipients. 62: The pharmaceutical composition ofclaim 59, comprising a further therapeutic agent. 63: The pharmaceuticalcomposition of claim 60, characterized in that the further therapeuticagent is an antagonist of epidermal-growth factor (EGF),fibroblast-growth factor (FGF), hepatocyte growth factor (HGF), tissuefactor (TF), protein C, protein S, platelet-derived growth factor(PDGF), heregulin, macrophage-stimulating protein (MSP) or vascularendothelial growth factor (VEGF), a receptor for epidermal-growth factor(EGF), fibroblast-growth factor (FGF), hepatocyte growth factor (HGF),tissue factor (TF), protein C, protein S, platelet-derived growth factor(PDGF), heregulin, macrophage-stimulating protein (MSP), vascularendothelial growth factor (VEGF), HER2 receptor, HER3 receptor, c-MET,or other receptor tyrosine kinase. 64: The pharmaceutical composition ofclaim 60, characterized in that the further therapeutic agent is anantibody directed against a cluster of differentiation antigen selectedfrom a group consisting of: CD3, CD14, CD19, CD20, CD22, CD25, CD28,CD30, CD33, CD36, CD40, CD44, CD52, CD55, CD59, CD56, CD70, CD79, CD80,CD103, CD134, CD137, CD138, and CD152. 65: A method of treating canceror autoimmune disease comprising administering to a patient the antibodyor epitope-binding fragment thereof according to claim 1, or a conjugateaccording to claim
 52. 66-71. (canceled) 72: A method of diagnosing acancer in a subject known to or suspected to have a cancer, said methodcomprising: a) Contacting cells of said patient with an antibody orepitope-binding fragment thereof according to claim 1, b) Measuring thebinding of said antibody or epitope-binding fragment thereof to saidcells, and c) comparing the expression in part b) with that of a normalreference subject or standard. 73-75. (canceled) 76: One or morepolynucleotides encoding an antibody or epitope-binding fragmentthereof, wherein the antibody or epitope-binding fragment thereofcomprises: a) a heavy chain variable region (V_(H)) comprising SEQ IDNO: 50 and a light chain variable region (V_(L)) comprising SEQ ID NO:38; b) a heavy chain variable region (V_(H)) comprising SEQ ID NO: 52and a light chain variable region (V_(L)) comprising SEQ ID NO: 40; c) aheavy chain variable region (V_(H)) comprising SEQ ID NO: 66 and a lightchain variable region (V_(L)) comprising SEQ ID NO: 62 or 64; d) a heavychain variable region (V_(H)) comprising SEQ ID NO: 56 and a light chainvariable region (V_(L)) comprising 44; e) a heavy chain variable region(V_(H)) comprising SEQ ID NO: 72 and a light chain variable region(V_(L)) comprising SEQ ID NO: 68 or 70; f) a heavy chain variable region(V_(H)) comprising SEQ ID NO: 60 and a light chain variable region(V_(L)) comprising
 48. 77. (canceled) 78: A recombinant vectorcomprising the one or more polynucleotides of claim
 76. 79: A host cellcomprising a vector of claim 78.